Modified antibody constant region

ABSTRACT

The present inventors succeeded in improving the antibody constant region to have increased stability under acid conditions, reduced heterogeneity originated from disulfide bonds in the hinge region, reduced heterogeneity originated from the H chain C terminus, and increased stability at high concentrations as well as in discovering novel constant region sequences having reduced Fcγ receptor-binding, while minimizing the generation of novel T-cell epitope peptides. As a result, the present inventors successfully discovered antibody constant regions with improved physicochemical properties (stability and homogeneity), immunogenicity, safety, and pharmacokinetics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/680,082, having a 371(c) date of Jun. 25, 2010, which is the NationalStage of International Application Serial No. PCT/JP2008/067483, filedon Sep. 26, 2008, which claims priority to Japanese Application SerialNo. 2007-250147, filed on Sep. 26, 2007. The contents of theapplications referenced above are hereby incorporated by reference intheir entireties in this application.

TECHNICAL FIELD

The present invention relates to antibody constant regions that haveimproved physicochemical properties (stability and homogeneity),immunogenicity (antigenicity), and safety, and/or half-life in plasma;and antibodies comprising the constant regions.

BACKGROUND ART

Antibodies are drawing attention as pharmaceuticals as they are highlystable in plasma (blood) and have few adverse effects. Of them, a numberof IgG-type antibody pharmaceuticals are available on the market andmany antibody pharmaceuticals are currently under development(Non-patent Documents 1 and 2).

Almost all antibody pharmaceuticals currently available on the marketare of the IgG1 subclass. IgG1 type antibodies are expected be useful asanti-cancer antibody pharmaceuticals since they can bind to Fcγ receptorand exert ADCC activity. However, binding of the Fc domain to Fcγreceptor, which is important for effector function such as ADCC, cancause unnecessary adverse effects, and thus it is preferable toeliminate such binding activity from antibody pharmaceuticals intendedfor neutralizing biological activity (Non-patent Document 3).Furthermore, since Fcγ receptor is expressed in antigen-presentingcells, molecules that bind to Fcγ receptor tend to be presented asantigens. It has been reported that immunogenicity is and can beenhanced by linking a protein or peptide to the Fc domain of IgG1(Non-patent Document 4 and Patent Document 1). Interaction between theantibody Fc domain and Fcγ receptor is thought to be a cause of theserious adverse effects encountered in phase-I clinical trials ofTGN1412 (Non-patent Document 5). Thus, binding to Fcγ receptor isconsidered unfavorable in antibody pharmaceuticals intended forneutralizing the biological activity of an antigen from the perspectiveof adverse effect and immunogenicity.

A method for impairing the binding to Fcγ receptor is to alter thesubtype of the IgG antibody from IgG1 to IgG2 or IgG4; however, thismethod cannot completely inhibit the binding (Non-patent Document 6).One of the methods reported for completely inhibiting the binding to Fcγreceptor is to artificially alter the Fc domain. For example, theeffector functions of anti-CD3 antibodies and anti-CD4 antibodies causeadverse effects. Thus, amino acids that are not present in the wild typesequence were introduced into the Fcγ -receptor-binding domain of Fc(Non-patent Documents 3 and 7), and clinical trials are currently beingconducted to assess anti-CD3 antibodies that do not bind to Fcγ receptorand anti-CD4 antibodies that have a mutated Fc domain (Non-patentDocuments 5 and 8). Alternatively, Fcγ receptor-nonbinding antibodiescan be prepared by altering the FcγR-binding domain of IgG1 (atpositions 233, 234, 235, 236, 327, 330, and 331 in the EU numberingsystem) to an IgG2 or IgG4 sequence (Non-patent Document 9 and PatentDocument 2). However, these molecules contain novel non-natural peptidesequences of nine to twelve amino acids, which may constitute a T-cellepitope peptide and thus pose immunogenicity risk. There is no previousreport on Fcγ receptor-nonbinding antibodies that have overcome theseproblems.

Meanwhile, physicochemical properties of antibody proteins, inparticular, homogeneity and stability, are very crucial in thedevelopment of antibody pharmaceuticals. For the IgG2 subtype,heterogeneity originated from disulfide bonds in the hinge region hasbeen reported (Non-patent Document 10 and Patent Document 3). It is noteasy to manufacture them as a pharmaceutical in large-scale whilemaintaining the objective substances/related substances relatedheterogeneity between productions. Thus, single substances are desirableas much as possible for antibody molecules developed as pharmaceuticals.

IgG2 and IgG4 are unstable under acidic conditions. IgG type antibodiesare in general exposed to acidic conditions in the purification processusing Protein A and the virus inactivation process. Thus, attention isneeded regarding the stability of IgG2 and IgG4 during these processes,and it is preferable that antibody molecules developed aspharmaceuticals are also stable under acidic conditions. Natural IgG2and IgG4, and Fcγ receptor-nonbinding antibodies derived from IgG2 orIgG4 (Non-patent Documents 6 and 7 and Patent Document 2) have suchproblems. It is desirable to solve these problems when developingantibodies into pharmaceuticals.

IgG1-type antibodies are relatively stable under acidic conditions, andthe degree of heterogeneity originated from disulfide bonds in the hingeregion is also lower in this type of antibodies. However, IgG1-typeantibodies are reported to undergo non-enzymatic peptide bond cleavagein the hinge region in solutions when they are stored as formulations,and Fab fragments are generated as impurities as a result (Non-patentDocument 11). It is desirable to overcome the generation of impuritywhen developing antibodies into pharmaceuticals.

Furthermore, for heterogeneity of the C-terminal sequence of anantibody, deletion of C-terminal amino acid lysine residue, andamidation of the C-terminal amino group due to deletion of both of thetwo C-terminal amino acids, glycine and lysine, have been reported(Non-patent Document 12). It is preferable to eliminate suchheterogeneity when developing antibodies into pharmaceuticals.

The constant region of an antibody pharmaceutical aimed for neutralizingan antigen preferably has a sequence that overcomes all the problemsdescribed above. However, a constant region that meets all therequirements has not been reported.

A preferred form of antibody pharmaceutical administration is thought tobe subcutaneous formulation in chronic autoimmune diseases and such.Low-cost, convenient antibody pharmaceuticals that can be administeredsubcutaneously in longer intervals can be provided by increasing thehalf-life of an antibody in the plasma to prolong its therapeutic effectand thereby reduce the amount of protein administered, and by conferringthe antibody with high stability so that high concentration formulationscan be prepared.

In general, it is necessary that subcutaneous formulations arehigh-concentration formulations. From the perspective of stability orsuch, the concentration limit of IgG-type antibody formulations is ingeneral thought to be about 100 mg/ml (Non-patent Document 13). Thus, itis a challenge to secure stability at high concentration. However, thereis no report published on the improvement of the stability of IgG athigh concentrations by introducing amino acid substitutions into itsconstant region. A method for prolonging the antibody half-life inplasma has been reported and it substitutes amino acids in the constantregion (Non-patent Documents 14 and 15); however, introduction ofnon-natural sequences into the constant region is not unpreferable fromthe perspective of immunogenicity risk.

As described above, when the purpose of an antibody pharmaceutical is toneutralize an antigen, it is preferable that all the problems describedabove have been overcome with regard to its constant-region sequence.However, a constant region that meets all the requirements has not beenreported. Thus, there are demands for antibody constant regions thathave overcome the problems described above.

Documents of related prior arts for the present invention are describedbelow.

-   [Non-patent Document 1] Janice M Reichert, Clark J Rosensweig, Laura    B Faden & Matthew C Dewitz. Monoclonal antibody successes in the    clinic. Nature Biotechnology (2005) 23, 1073-1078-   [Non-patent Document 2] Pavlou A K, Belsey M J. The therapeutic    antibodies market to 2008. Eur. J. Pharm. Biopharm. 2005 April;    59(3):389-96-   [Non-patent Document 3] Reddy M P, Kinney C A, Chaikin M A, Payne A,    Fishman-Lobell J, Tsui P, Dal Monte P R, Doyle M L, Brigham-Burke M    R, Anderson D, Reff M, Newman R, Hanna N, Sweet R W, Truneh A.    Elimination of Fc receptor-dependent effector functions of a    modified IgG4 monoclonal antibody to human CD4. J. Immunol. 2000    Feb. 15; 164(4):1925-33-   [Non-patent Document 4] Guyre P M, Graziano R F, Goldstein J,    Wallace P K, Morganelli P M, Wardwell K, Howell A L. Increased    potency of Fc-receptor-targeted antigens. Cancer Immunol.    Immunother. 1997 November-December; 45(3-4):146-8-   [Non-patent Document 5] Strand V, Kimberly R, Isaacs J D. Biologic    therapies in rheumatology: lessons learned, future directions. Nat.    Rev. Drug Discov. 2007 January; 6(1):75-92-   [Non-patent Document 6] Gessner J E, Heiken H, Tamm A, Schmidt R E.    The IgG Fc receptor family. Ann. Hematol. 1998 June; 76(6):231-48-   [Non-patent Document 7] Cole M S, Anasetti C, Tso J Y. Human IgG2    variants of chimeric anti-CD3 are nonmitogenic to T cells. J.    Immunol. 1997 Oct. 1; 159(7):3613-21-   [Non-patent Document 8] Chau L A, Tso J Y, Melrose J, Madrenas J.    HuM291(Nuvion), a humanized Fc receptor-nonbinding antibody against    CD3, anergizes peripheral blood T cells as partial agonist of the T    cell receptor. Transplantation 2001 Apr. 15; 71(7):941-50-   [Non-patent Document 9] Armour K L, Clark M R, Hadley A G,    Williamson L M. Recombinant human IgG molecules lacking Fcgamma    receptor I binding and monocyte triggering activities. Eur. J.    Immunol. 1999 August; 29(8):2613-24-   [Non-patent Document 10] Chu G C, Chelius D, Xiao G, Khor H K,    Coulibaly S, Bondarenko P V. Accumulation of Succinimide in a    Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under    Elevated Temperatures. Pharm. Res. 2007 Mar. 24; 24(6):1145-56-   [Non-patent Document 11] A J Cordoba, B J Shyong, D Breen, R J    Harris. Nonenzymatic hinge region fragmentation of antibodies in    solution. J. Chromatogr. B. Anal. Technol. Biomed. Life Sci. (2005)    818, 115-121-   [Non-patent Document 12] Johnson K A, Paisley-Flango K, Tangarone B    S, Porter T J, Rouse J C. Cation exchange-HPLC and mass spectrometry    reveal C-terminal amidation of an IgG1 heavy chain. Anal. Biochem.    2007 Jan. 1; 360(1):75-83-   [Non-patent Document 13] Shire S J, Shahrokh Z, Liu J. Challenges in    the development of high protein concentration formulations. J.    Pharm. Sci. 2004 June; 93(6):1390-402-   [Non-patent Document 14] Hinton P R, Xiong J M, Johlfs M G, Tang M    T, Keller S, Tsurushita N. An engineered human IgG1 antibody with    longer serum half-life. J. Immunol. 2006 Jan. 1; 176(1):346-56-   [Non-patent Document 15] Ghetie V, Popov S, Borvak J, Radu C,    Matesoi D, Medesan C, Ober R J, Ward E S. Increasing the serum    persistence of an IgG fragment by random mutagenesis. Nat.    Biotechnol. 1997 July; 15(7):637-40-   [Patent Document 1] US 20050261229A1-   [Patent Document 2] WO 99/58572-   [Patent Document 3] US 2006/0194280

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide antibody constantregions that have improved physicochemical properties (stability andhomogeneity), immunogenicity, safety, and pharmacokinetics (retention inplasma (blood)) by amino acid alteration.

Means for Solving the Problems

The present inventors conducted dedicated studies to generate antibodyconstant regions that are improved through alternation of their aminoacid sequences and have improved physicochemical properties (stabilityand homogeneity), immunogenicity, and safety, and pharmacokinetics. As aresult, the present inventors successfully improved antibody constantregion to have increased stability under acid conditions, reducedheterogeneity originated from disulfide bonds in the hinge region,reduced heterogeneity originated from the H-chain C terminus, andincreased stability at high concentrations, as well as discovered novelconstant region sequences having reduced Fcγ receptor-binding activity,while minimizing the generation of novel T-cell epitope peptides.

The present invention relates to antibody constant regions that aresuperior in terms of safety, immunogenicity risk, physicochemicalproperties (stability and homogeneity), and pharmacokinetics a throughimprovement by amino acid alteration; antibodies comprising suchantibody constant region; pharmaceutical compositions comprising suchantibody; and methods for producing them. More specifically, the presentinvention provides:

-   [1] a human antibody constant region of any one of:-   (a) a human antibody constant region that comprises deletions of    both Gly at position 329 (position 446 in the EU numbering system,    see sequences of proteins of immunological interest, NIH Publication    No.91-3242) and Lys at position 330 (position 447 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 1;-   (b) a human antibody constant region that comprises deletions of    both Gly at position 325 (position 446 in the EU numbering system)    and Lys at position 326 (position 447 in the EU numbering system) in    the amino acid sequence of SEQ ID NO: 2; and-   (c) a human antibody constant region that comprises deletions of    both Gly at position 326 (position 446 in the EU numbering system)    and Lys at position 327 (position 447 in the EU numbering system) in    the amino acid sequence of SEQ ID NO: 3;-   [2] an IgG2 constant region in which the amino acids at positions    209 (position 330 in the EU numbering system), 210 (position 331 in    the EU numbering system), and 218 (position 339 in the EU numbering    system) in the amino acid sequence of SEQ ID NO: 2 have been    substituted with other amino acids;-   [3] an IgG2 constant region in which the amino acid at position 276    (position 397 in the EU numbering system) in the amino acid sequence    of SEQ ID NO: 2 has been substituted with another amino acid;-   [4] an IgG2 constant region in which the amino acids at positions 14    (position 131 in the EU numbering system), 102 (position 219 in the    EU numbering system), and/or 16 (position 133 in the EU numbering    system) in the amino acid sequence of SEQ ID NO: 2 have been    substituted with another amino acid;-   [5] the IgG2 constant region of [4], in which the amino acids at    positions 20 (position 137 in the EU numbering system) and 21    (position 138 in the EU numbering system) in the amino acid sequence    of SEQ ID NO: 2 have been substituted with other amino acids;-   [6] an IgG2 constant region in which His at position 147 (position    268 in the EU numbering system), Arg at position 234 (position 355    in the EU numbering system), and/or Gln at position 298 (position    419 in the EU numbering system) in the amino acid sequence of SEQ ID    NO: 2 have been substituted with other amino acids;-   [7] an IgG2 constant region in which the amino acids at positions    209 (position 330 in the EU numbering system), 210 (position 331 in    the EU numbering system), 218 (position 339 in the EU numbering    system), 276 (position 397 in the EU numbering system), 14 (position    131 in the EU numbering system), 16 (position 133 in the EU    numbering system), 102 (position 219 in the EU numbering system), 20    (position 137 in the EU numbering system), and 21 (position 138 in    the EU numbering system) in the amino acid sequence of SEQ ID NO: 2    have been substituted with other amino acids;-   [8] the IgG2 constant region of [7], which further comprises    deletions of both Gly at position 325 (position 446 in the EU    numbering system) and Lys at position 326 (position 447 in the EU    numbering system);-   [9] an IgG2 constant region in which the amino acids at positions    276 (position 397 in the EU numbering system), 14 (position 131 in    the EU numbering system), 16 (position 133 in the EU numbering    system), 102 (position 219 in the EU numbering system), 20 (position    137 in the EU numbering system), and 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 have    been substituted with other amino acids;-   [10] the IgG2 constant region of [9], which further comprises    deletions of both Gly at position 325 (position 446 in the EU    numbering system) and Lys at position 326 (position 447 in the EU    numbering system);-   [11] an IgG2 constant region in which Cys at position 14 (position    131 in the EU numbering system), Arg at position 16 (position 133 in    the EU numbering system), Cys at position 102 (position 219 in the    EU numbering system), Glu at position 20 (position 137 in the EU    numbering system), Ser at position 21 (position 138 in the EU    numbering system), His at position 147 (position 268 in the EU    numbering system), Arg at position 234 (position 355 in the EU    numbering system), and Gln at position 298 (position 419 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 have    been substituted with other amino acids;-   [12] the IgG2 constant region of [11], which further comprises    deletions of both Gly at position 325 (position 446 in the EU    numbering system) and Lys at position 326 (position 447 in the EU    numbering system);-   [13] an IgG2 constant region in which Cys at position 14 (position    131 in the EU numbering system), Arg at position 16 (position 133 in    the EU numbering system), Cys at position 102 (position 219 in the    EU numbering system), Glu at position 20 (position 137 in the EU    numbering system), Ser at position 21 (position 138 in the EU    numbering system), His at position 147 (position 268 in the EU    numbering system), Arg at position 234 (position 355 in the EU    numbering system), Gln at position 298 (position 419 in the EU    numbering system), and Asn at position 313 (position 434 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 have    been substituted with other amino acids;-   [14] the IgG2 constant region of [13], which further comprises    deletions of both Gly at position 325 (position 446 in the EU    numbering system) and Lys at position 326 (position 447 in the EU    numbering system);-   [15] an IgG4 constant region in which the amino acid at position 289    (position 409 in the EU numbering system) in the amino acid sequence    of SEQ ID NO: 3 has been substituted with another amino acid;-   [16] an IgG4 constant region in which the amino acids at position    289 (position 409 in the EU numbering system), positions 14, 16, 20,    21, 97, 100, 102, 103, 104, and 105 (positions 131, 133, 137, 138,    214, 217, 219, 220, 221, and 222 in the EU numbering system,    respectively), and positions 113, 114, and 115 (positions 233, 234,    and 235 in the EU numbering system, respectively), have been    substituted with other amino acids, and the amino acid at position    116 (position 236 in the EU numbering system) has been deleted from    the amino acid sequence of SEQ ID NO: 3;-   [17] the IgG4 constant region of [16], which further comprises    deletions of both Gly at position 326 (position 446 in the EU    numbering system) and Lys at position 327 (position 447 in the EU    numbering system);-   [18] an IgG1 constant region in which Asn at position 317 (position    434 in the EU numbering system) in the amino acid sequence of SEQ ID    NO: 1 has been substituted with another amino acid;-   [19] the IgG1 constant region of [18], which further comprises    deletions of both Gly at position 329 (position 446 in the EU    numbering system) and Lys at position 330 (position 447 in the EU    numbering system);-   [20] an IgG2 constant region in which Ala at position 209 (position    330 in the EU numbering system), Pro at position 210 (position 331    in the EU numbering system), Thr at position 218 (position 339 in    the EU numbering system), Cys at position 14 (position 131 in the EU    numbering system), Arg at position 16 (position 133 in the EU    numbering system), Cys at position 102 (position 219 in the EU    numbering system), Glu at position 20 (position 137 in the EU    numbering system), and Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 have    been substituted with other amino acids;-   [21] the IgG2 constant region of [20], which further comprises    deletions of both Gly at position 325 (position 446 in the EU    numbering system) and Lys at position 326 (position 447 in the EU    numbering system);-   [22] an IgG2 constant region in which Cys at position 14 (position    131 in the EU numbering system), Arg at position 16 (position 133 in    the EU numbering system), Cys at position 102 (position 219 in the    EU numbering system), Glu at position 20 (position 137 in the EU    numbering system), and Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 have    been substituted with other amino acids;-   [23] the IgG2 constant region of [22], which further comprises    deletions of both Gly at position 325 (position 446 in the EU    numbering system) and Lys at position 326 (position 447 in the EU    numbering system);-   [24] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 5;-   [25] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 7;-   [26] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 9;-   [27] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 35;-   [28] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 36;-   [29] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 37;-   [30] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 43;-   [31] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 57 (M40ΔGK);-   [32] a human antibody constant region comprising the amino acid    sequence of SEQ ID NO: 55 (M86ΔGK);-   [33] an antibody comprising the constant region of any one of [1] to    [32];-   [34] an anti-IL-6 receptor antibody comprising the constant region    of any one of [1] to [32]; and-   [35] a pharmaceutical composition comprising the constant region of    any one of [1] to [32].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the result of using gel filtrationchromatography to analyze the content of aggregates in WT-IgG1, WT-IgG2,WT-IgG4, IgG2-M397V, and IgG4-R409K purified by hydrochloric acidelution.

FIG. 2 is a diagram showing the result of cation exchange chromatography(IEC) analysis of WT-IgG1, WT-IgG2, and WT-IgG4.

FIG. 3 is a diagram showing predicted disulfide bonding in the hingeregion of WT-IgG2.

FIG. 4 is a diagram showing predicted disulfide bonding in the hingeregion of IgG2-SKSC.

FIG. 5 is a diagram showing the result of cation exchange chromatography(IEC) analysis of WT-IgG2 and IgG2-SKSC.

FIG. 6 is a diagram showing the result of cation exchange chromatography(IEC) analysis of humanized PM-1 antibody, H chain C-terminal ΔKantibody, and H chain C-terminal ΔGK antibody.

FIG. 7 shows comparison of the amounts WT-IgG1, WT-IgG2, WT-IgG4,WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK bound to FcγRI.

FIG. 8 is a graph showing comparison of the amounts WT-IgG1, WT-IgG2,WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK bound to FcγRIIa.

FIG. 9 is a graph showing comparison of the amounts WT-IgG1, WT-IgG2,WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK bound to FcγRIIb.

FIG. 10 is a graph showing comparison of the amounts WT-IgG1, WT-IgG2,WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK bound to FcγRIIIa (Val).

FIG. 11 is a graph showing the increase of aggregation in a stabilitytest for WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK at highconcentrations.

FIG. 12 is a graph showing the increase of Fab fragments in a stabilitytest for WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK at highconcentrations.

FIG. 13 is a diagram showing the result of cation exchangechromatography (IEC) analysis of WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK.

FIG. 14 is a graph showing the time courses of plasma concentrations ofWT-IgG1 and WT-M14 after intravenous administration to human FcRntransgenic mice.

FIG. 15 is a graph showing the time courses of plasma concentrations ofWT-IgG1, WT-M44, WT-M58, and WT-M73 after intravenous administration tohuman FcRn transgenic mice.

FIG. 16 is a diagram showing a cation exchange chromatography-basedassessment of the effect on heterogeneity by the constant region of antiIL-6 receptor antibodies WT and F2H/L39, anti-IL-31 receptor antibodyH0L0 , and anti-RANKL antibody DNS.

FIG. 17 is a diagram showing a cation exchange chromatography-basedassessment of the effect on heterogeneity by the CH1 domain cysteine ofanti IL-6 receptor antibodies WT and F2H/L39.

FIG. 18 is a diagram showing a DSC-based assessment of the effect ondenaturation peak by the CH1 domain cysteine of anti IL-6 receptorantibody WT and F2H/L39.

FIG. 19 is a graph showing the activities of TOCILIZUMAB, the control,and Fv5-M83 to neutralize BaF/g130.

FIG. 20 is a graph showing the activities of TOCILIZUMAB, Fv3-M73, andFv4-M73 to neutralize BaF/gp130.

FIG. 21 is a graph showing the plasma concentration time courses ofTOCILIZUMAB, the control, Fv3-M73, Fv4-M73, and Fv5-M83 in cynomolgusmonkeys after intravenous administration.

FIG. 22 is a graph showing the time courses of CRP concentration incynomolgus monkeys after intravenous administration of TOCILIZUMAB, thecontrol, Fv3-M73, Fv4-M73, or Fv5-M83.

FIG. 23 is a graph showing the time courses of concentration of freesoluble IL-6 receptor in cynomolgus monkeys after intravenousadministration of TOCILIZUMAB, the control, Fv3-M73, Fv4-M73, orFv5-M83.

FIG. 24 is a graph showing the time courses of plasma concentrations ofWT-IgG1, WT-M14, and WT-M58 after intravenous administration to humanFcRn transgenic mice.

MODE FOR CARRYING OUT THE INVENTION

The present invention provides antibody constant regions whosephysicochemical properties (stability and homogeneity), immunogenicity,safety, and/or pharmacokinetics have been improved by altering the aminoacid sequence of an antibody constant region; antibodies comprising suchconstant region; pharmaceutical compositions comprising such antibody;and methods for producing them.

Herein, the constant region refers to IgG1, IgG2, or IgG4 type constantregion. The antibody constant region is preferably a human antibodyconstant region. The amino acid sequences of human IgG1, IgG2, and IgG4constant regions are known (human IgG1 constant region, SEQ ID NO: 1;human IgG2 constant region, SEQ ID NO: 2; and human IgG4 constantregion, SEQ ID NO: 3). The amino acid substitution-containing antibodyconstant regions of the present invention may comprise other amino acidsubstitutions or modifications as long as they comprise the amino acidsubstitutions of the present invention. Therefore, IgG2 constant regionscomprising the amino acid substitutions of the present invention in theIgG2 constant region comprising the amino acid sequence of SEQ ID NO: 2include IgG2 constant regions that comprise one or more amino acidsubstitutions and/or modifications in the amino acid sequence of SEQ IDNO: 2 and further comprise the amino acid substitutions of the presentinvention, as well as IgG2 constant regions that comprise the amino acidsubstitutions of the present invention and further comprise one or moreamino acid substitutions and/or modifications. The same applies to IgG1constant regions comprising the amino acid sequence of SEQ ID NO: 1 andIgG4 constant regions comprising the amino acid sequence of SEQ ID NO:3. The sequence of human IgG4 constant region has been altered toimprove the stability of the hinge region (Mol. Immunol. 1993 January;30(1):105-8). Furthermore, the sugar chain at position 297 in the EUnumbering system may be of any sugar-chain structure, or there may notbe any sugar chain linked at this site (for example, can be producedwith E. coli).

<IgG2 Having Altered Amino Acids>

The present invention provides IgG2 constant regions with an improvedstability under acid conditions.

More specifically, the present invention provides IgG2 constant regionsin which Met at position 276 (position 397 in the EU numbering system)in the amino acid sequence of SEQ ID NO: 2 has been substituted withanother amino acid. The type of amino acid after substitution is notparticularly limited; however, substitution to Val is preferred. Theantibody stability under acidic conditions can be improved bysubstituting Met at position 276 (position 397 in the EU numberingsystem) in the amino acid sequence of SEQ ID NO: 2 with another aminoacid.

The IgG2 constant regions provided by the present invention, which havean improved stability under acid conditions, may also have other aminoacid substitutions, deletions, additions, and/or insertions, as long asthey have at least the amino acid substitution described above.

The present invention provides IgG2 constant regions with reducedheterogeneity of hinge region.

More specifically, the present invention provides IgG2 constant regionsin which Cys at position 14 (position 131 in the EU numbering system),Arg at position 16 (position 133 in the EU numbering system), and/or Cysat position 102 (position 219 in the EU numbering system) in the aminoacid sequence of SEQ ID NO: 2 have been substituted with other aminoacids. The type of amino acid after substitution is not particularlylimited; however, substitutions of Ser for Cys at position 14 (position131 in the EU numbering system), Lys for Arg at position 16 (position133 in the EU numbering system), and Ser for Cys at position 102(position 219 in the EU numbering system) (IgG2-SKSC) are preferred.

These substitutions can reduce the heterogeneity originated from thehinge region of IgG2. The IgG2 constant regions of the present inventioncomprising amino acid substitutions include IgG2 constant regionscomprising at least one of the three types of amino acid substitutionsdescribed above; however, the IgG2 constant regions preferably comprisesubstitutions of Cys at position 14 and Cys at position 102 with otheramino acids or all three types of the amino acid substitutions describedabove.

The IgG2 constant regions provided by the present invention, which havereduced heterogeneity, may also have other amino acid substitutions,deletions, additions, and/or insertions, as long as they have at leastthe amino acid substitution described above.

For example, mutating Cys at position 14 and Arg at position 16 in anIgG2 constant region comprising the amino acid sequence of SEQ ID NO: 2may generate non-natural, novel peptide sequences of nine to twelveamino acids, which can become T-cell epitope peptides, and thus generateimmunogenicity risk. Even with the introduction of the amino acidsubstitutions described above, the generation of non-natural T-cellepitope peptides can be avoided by substituting Glu at position 20(position 137 in the EU numbering system) and Ser at position 21(position 138 in the EU numbering system) with other amino acids. Thetype of amino acid after substitution is not particularly limited;however, substitutions of Gly for Glu at position 20 and Gly for Ser atposition 21 are preferred.

The present invention also provides IgG2 constant regions with reducedFcγ receptor-binding activity.

More specifically, the present invention also provides IgG2 constantregions comprising an amino acid sequence in which Ala at position 209(EU330), Pro at position 210 (EU331), and/or Thr at position 218 (EU339)of the amino acid sequence of SEQ ID NO: 2 have been substituted withSer, Ser, and Ala, respectively. The substitutions for Ala at position209 (EU330) and for Pro at position 210 (EU331) have already beenreported to enable the impairment of the Fcγ receptor binding (Eur. J.Immunol. 1999 August; 29(8):2613-24). From the perspective ofimmunogenicity risk, however, these alterations are not preferredbecause they result in generation of non-human derived peptides that canbecome T-cell epitopes. However, the Fcγ receptor binding of IgG2 can bereduced by substituting Ala for Thr at position 218 (EU339) at the sametime, and the 9-12 amino acid peptides which can become T-cell epitopesare derived from human only.

The IgG2 constant regions of the present invention comprising amino acidsubstitutions comprise at least one of the three types of amino acidsubstitutions described above; however, the IgG2 constant regionspreferably comprise all three types of the amino acid substitutionsdescribed above. In a preferred embodiment, the IgG2 constant regions ofthe present invention comprising amino acid substitutions include IgG2constant regions comprising an amino acid sequence in which Ala atposition 209 (EU330), Pro at position 210 (EU331), and Thr at position218 (EU339) in the amino acid sequence of SEQ ID NO: 2 have beensubstituted with Ser, Ser, and Ala, respectively.

The IgG2 constant regions provided by the present invention, which havereduced Fcγ receptor-binding activity, may also have other amino acidsubstitutions, deletions, additions, and/or insertions, as long as theyhave at least the amino acid substitution described above.

The present invention provides IgG2 constant regions with reducedC-terminal heterogeneity.

More specifically, the present invention provides IgG2 constant regionscomprising an amino acid sequence in which Gly at position 325 (position446 in the EU numbering system) and Lys at position 326 (position 447 inthe EU numbering system) have been deleted in the amino acid sequence ofSEQ ID NO: 2. The heterogeneity originated from the C terminus ofantibody H chain can be reduced only when both of the amino acids aredeleted.

The IgG2 constant regions provided by the present invention, which havereduced C-terminal heterogeneity, may also have other amino acidsubstitutions, deletions, additions, and/or insertions, as long as theyhave at least the amino acid substitution described above.

The present invention further provides IgG2 constant regions withimproved pharmacokinetics.

Specifically, the present invention provides IgG2 constant regions inwhich His at position 147 (position 268 in the EU numbering system), Argat position 234 (position 355 in the EU numbering system), and Gln atposition 298 (position 419 in the EU numbering system) in the amino acidsequence of SEQ ID NO: 2 have been substituted with other amino acids.These amino acid substitutions enable to improve antibodypharmacokinetics. The type of amino acid after substitution is notparticularly limited; however, substitutions of Gln for His at position147 (position 268 in the EU numbering system), Gln for Arg at position234 (position 355 in the EU numbering system), and Glu for Gln atposition 298 (position 419 in the EU numbering system) are preferred.The IgG2 constant regions with amino acid substitutions of the presentinvention include IgG2 constant regions comprising at least one of thethree types of the amino acid substitutions described above; however,the IgG2 constant regions preferably comprise all three types of theamino acid substitutions described above.

Below is a preferred embodiment of IgG2 of the present invention, whichhas improved stability under acidic conditions, reduced heterogeneity inthe hinge region, and/or reduced Fcγ receptor-binding activity.

Antibodies comprising an IgG2 constant region comprising an amino acidsequence in which Ala at position 209, Pro at position 210, Thr atposition 218, Met at position 276, Cys at position 14, Arg at position16, Cys at position 102, Glu at position 20, and Ser at position 21 inthe amino acid sequence of SEQ ID NO: 2 have been substituted with otheramino acids.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Ala at position 209 (position 330 inthe EU numbering system), Ser for Pro at position 210 (position 331 inthe EU numbering system), Ala for Thr at position 218 (position 339 inthe EU numbering system), Val for Met at position 276 (position 397 inthe EU numbering system), Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Ser for Cys at position 102 (position 219 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), and Gly for Ser at position 21 (position 138in the EU numbering system) are preferred.

Such IgG2 constant regions include, for example, IgG2 constant regionscomprising the amino acid sequence of SEQ ID NO: 4 (M14).

In another preferred embodiment, IgG2 constant regions of the presentinvention include IgG2 constant regions resulting from the deletion ofGly at position 325 and Lys at position 326 in the above-described IgG2constant regions to reduce C-terminal heterogeneity. Such antibodiesinclude, for example, IgG2 that comprises a constant region comprisingthe amino acid sequence of SEQ ID NO: 5 (M14ΔGK).

Below is a preferred embodiment of IgG2 of the present invention, whichhas reduced heterogeneity in the hinge region and/or reduced Fcγreceptor-binding activity.

Antibodies comprising an IgG2 constant region comprising an amino acidsequence in which Ala at position 209, Pro at position 210, Thr atposition 218, Cys at position 14, Arg at position 16, Cys at position102, Glu at position 20, and Ser at position 21 in the amino acidsequence of SEQ ID NO: 2 have been substituted with other amino acids.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Ala at position 209 (position 330 inthe EU numbering system), Ser for Pro at position 210 (position 331 inthe EU numbering system), Ala for Thr at position 218 (position 339 inthe EU numbering system), Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Ser for Cys at position 102 (position 219 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), and Gly for Ser at position 21 (position 138in the EU numbering system) are preferred.

Such IgG2 constant regions include, for example, IgG2 constant regionscomprising the amino acid sequence of SEQ ID NO: 54 (M86).

In another preferred embodiment, IgG2 constant regions of the presentinvention include IgG2 constant regions resulting from the deletion ofGly at position 325 and Lys at position 326 in the above-described IgG2constant regions to reduce C-terminal heterogeneity. Such antibodiesinclude, for example, IgG2 that comprises a constant region comprisingthe amino acid sequence of SEQ ID NO: 55 (M86ΔGK).

Below is another preferred embodiment of the IgG2 constant regions ofthe present invention, which have improved stability under acidicconditions and reduced heterogeneity in the hinge region.

IgG2 constant regions comprising an amino acid sequence in which Met atposition 276, Cys at position 14, Arg at position 16, Cys at position102, Glu at position 20, and Ser at position 21 in the amino acidsequence of SEQ ID NO: 2 have been substituted with other amino acids.

The type of amino acid after substitution is not particularly limited;however, substitutions of Val for Met at position 276 (position 397 inthe EU numbering system), Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Ser for Cys at position 102 (position 219 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), and Gly for Ser at position 21 (position 138in the EU numbering system) are preferred.

Such IgG2 constant regions include, for example, IgG2 constant regionscomprising the amino acid sequence of SEQ ID NO: 6 (M31).

In another preferred embodiment, the IgG2 constant regions of thepresent invention include IgG2 constant regions further comprising thedeletion of Gly at position 325 and Lys at position 326 in theabove-described IgG2 constant regions. Such antibodies include, forexample, IgG2 constant regions comprising the amino acid sequence of SEQID NO: 7 (M31ΔGK).

Below is another preferred embodiment of the IgG2 constant regions ofthe present invention, which have reduced heterogeneity in the hingeregion.

IgG2 constant regions comprising an amino acid sequence in which Cys atposition 14, Arg at position 16, Cys at position 102, Glu at position20, and Ser at position 21 in the amino acid sequence of SEQ ID NO: 2have been substituted with other amino acids.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Ser for Cys at position 102 (position 219 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), and Gly for Ser at position 21 (position 138in the EU numbering system) are preferred.

Such IgG2 constant regions include, for example, IgG2 constant regionscomprising the amino acid sequence of SEQ ID NO: 56 (M40).

In another preferred embodiment, the IgG2 constant regions of thepresent invention include IgG2 constant regions further comprising thedeletion of Gly at position 325 and Lys at position 326 in theabove-described IgG2 constant regions. Such antibodies include, forexample, IgG2 constant regions comprising the amino acid sequence of SEQID NO: 57 (M40ΔGK).

The present invention provides IgG2 constant regions comprising an aminoacid sequence in which Cys at position 14 (position 131 in the EUnumbering system), Arg at position 16 (position 133 in the EU numberingsystem), Cys at position 102 (position 219 in the EU numbering system),Glu at position 20 (position 137 in the EU numbering system), Ser atposition 21 (position 138 in the EU numbering system), His at position147 (position 268 in the EU numbering system), Arg at position 234(position 355 in the EU numbering system), and Gln at position 298(position 419 in the EU numbering system) have been substituted withother amino acids, and simultaneously Gly at position 325 (position 446in the EU numbering system) and Lys at position 326 (position 447 in theEU numbering system) have been deleted in the amino acid sequence of SEQID NO: 2.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Cys at position 14, Lys for Arg atposition 16, Ser for Cys at position 102, Gly for Glu at position 20,Gly for Ser at position 21, Gln for His at position 147, Gln for Arg atposition 234, and Glu for Gln at position 298 are preferred.

Specifically, the present invention provides an antibody constant regioncomprising the amino acid sequence of SEQ ID NO: 35 (M58).

The present invention provides IgG2 constant regions comprising an aminoacid sequence in which Cys at position 14 (position 131 in the EUnumbering system), Arg at position 16 (position 133 in the EU numberingsystem), Cys at position 102 (position 219 in the EU numbering system),Glu at position 20 (position 137 in the EU numbering system), Ser atposition 21 (position 138 in the EU numbering system), His at position147 (position 268 in the EU numbering system), Arg at position 234(position 355 in the EU numbering system), Gln at position 298 (position419 in the EU numbering system), and Asn at position 313 (position 434in the EU numbering system) have been substituted with other aminoacids, and simultaneously Gly at position 325 (position 446 in the EUnumbering system) and Lys at position 326 (position 447 in the EUnumbering system) have been deleted in the amino acid sequence of SEQ IDNO: 2.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Cys at position 14, Lys for Arg atposition 16, Ser for Cys at position 102, Gly for Glu at position 20,Gly for Ser at position 21, Gln for His at position 147, Gln for Arg atposition 234, Glu for Gln at position 298, and Ala for Asn at position313 are preferred.

Specifically, the present invention provides an antibody constant regioncomprising the amino acid sequence of SEQ ID NO: 37 (M73).

These antibody constant regions have been optimized to have reduced Fcγreceptor binding activity, reduced immunogenicity risk, improvedstability under acidic conditions, reduced heterogeneity, improvedpharmacokinetics, and/or higher stability in preparations in comparisonwith the IgG1 constant region.

<IgG4 Having Altered Amino Acids>

The present invention provides IgG4 constant regions that are stable atacidic conditions.

More specifically, the present invention provides IgG4 constant regionscomprising an amino acid sequence in which Arg at position 289 (position409 in the EU numbering system) of the amino acid sequence of SEQ ID NO:3 has been substituted with another amino acid. The type of amino acidafter substitution is not particularly limited; however, substitution toLys is preferred. The antibody stability under acidic conditions can beimproved by substituting Arg at position 277 (position 409 in the EUnumbering system) in the amino acid sequence of SEQ ID NO: 3 withanother amino acid.

The IgG4 constant regions provided by the present invention, which havean improved stability under acidic conditions, may also have other aminoacid substitutions, deletions, additions, and/or insertions, as long asthey have at least the amino acid substitution described above.

The present invention provides IgG4 constant regions with reducedC-terminal heterogeneity.

The present invention provides IgG4 constant regions in which Gly atposition 326 (position 446 in the EU numbering system) and Lys atposition 327 (position 447 in the EU numbering system) have been deletedin the IgG4 constant region comprising the amino acid sequence of SEQ IDNO: 3. The heterogeneity originated from the C terminus of antibody Hchain can be reduced only when both of the amino acids are deleted.

The IgG4 constant regions provided by the present invention, which havereduced C-terminal heterogeneity, may also have other amino acidsubstitutions, deletions, additions, and/or insertions, as long as theyhave at least the amino acid substitution described above.

Another preferred embodiment of IgG4 of the present invention, which hasimproved stability under acidic conditions, reduced heterogeneity in thehinge region, and/or reduced Fcγ receptor-binding activity, includesIgG4 comprising the constant region described below.

IgG4 constant regions comprising an amino acid sequence in which Cys atposition 14, Arg at position 16, Glu at position 20, Ser at position 21,Arg at position 97, Ser at position 100, Tyr at position 102, Gly atposition 103, Pro at position 104, Pro at position 105, Glu at position113, Phe at position 114, Leu at position 115, and Arg at position 289have been substituted with other amino acids, and simultaneously Gly atposition 116 has been deleted in the amino acid sequence of SEQ ID NO:3.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), Gly for Ser at position 21 (position 138 inthe EU numbering system), Thr for Arg at position 97 (position 214 inthe EU numbering system), Arg for Ser at position 100 (position 217 inthe EU numbering system), Ser for Tyr at position 102 (position 219 inthe EU numbering system), Cys for Gly at position 103 (position 220 inthe EU numbering system), Val for Pro at position 104 (position 221 inthe EU numbering system), Glu for Pro at position 105 (position 222 inthe EU numbering system), Pro for Glu at position 113 (position 233 inthe EU numbering system), Val for Phe at position 114 (position 234 inthe EU numbering system), Ala for Leu at position 115 (position 235 inthe EU numbering system), and Lys for Arg at position 289 (position 409in the EU numbering system) are preferred.

Such IgG4 constant regions include, for example, IgG4 constant regionscomprising the amino acid sequence of SEQ ID NO: 8 (M11).

In another preferred embodiment, the IgG4 constant regions of thepresent invention include IgG4 constant regions further comprising thedeletion of Gly at position 325 (position 446 in the EU numberingsystem) and Lys at position 326 (position 447 in the EU numberingsystem) in the above-described IgG4 constant region. Such antibodiesinclude, for example, IgG4 constant regions comprising the amino acidsequence of SEQ ID NO: 9 (M11ΔGK).

<IgG1 Having Altered Amino Acids>

The present invention provides IgG1 constant regions with reducedC-terminal heterogeneity.

More specifically, the present invention provides IgG1 constant regionshaving the deletion of Gly at position 329 (position 446 in the EUnumbering system) and Lys at position 330 (position 447 in the EUnumbering system) in the IgG1 constant region comprising the amino acidsequence of SEQ ID NO: 1. The heterogeneity originated from the H-chainC terminus of an antibody can be reduced only when both of the aminoacids are deleted.

The present invention provides IgG1 constant regions with improvedpharmacokinetics.

The present invention provides IgG1 constant regions comprising an aminoacid sequence in which Asn at position 317 (position 434 in the EUnumbering system) in the amino acid sequence of SEQ ID NO: 1 has beensubstituted with another amino acid. The type of amino acid aftersubstitution is not particularly limited; however, substitution to Alais preferred.

The present invention provides a constant region having the deletion ofGly at position 329 and Lys at position 330 in the amino acid sequenceof SEQ ID NO: 36. More specifically, the present invention provides anantibody constant region comprising the amino acid sequence of SEQ IDNO: 43 (M83).

The IgG1 constant regions provided by the present invention, which havereduced C-terminal heterogeneity, may also have other amino acidsubstitutions, deletions, additions, and/or insertions, as long as theyhave at least the amino acid deletions described above.

The present invention also provides antibodies comprising any one of theantibody constant regions described above. The type and origin ofantibodies of the present invention are not particularly limited, aslong as they comprise the antibody constant region described above, andcan be any antibodies.

The antibodies of the present invention also include modified productsof antibodies comprising any of the amino acid substitutions describedabove. The origin of antibodies is not particularly limited. Theantibodies include human, mouse, rat, and rabbit antibodies. Theantibodies of the present invention may be chimeric, humanized, fullyhumanized antibodies, or such. In a preferred embodiment, the antibodiesof the present invention are humanized antibodies.

Alternatively, the antibody constant regions described above and/orantibody molecules comprising an antibody constant region describedabove can be linked as a form of Fc fusion molecule to antibody-likebinding molecule (scaffold molecules), bioactive peptides, bindingpeptides, or such.

The antibodies of the present invention also include modificationproducts of an antibody comprising any one of the constant regionsdescribed above.

Such antibody modification products include, for example, antibodieslinked with various molecules such as polyethylene glycol (PEG) andcytotoxic substances. Such antibody modification products can beobtained by chemically modifying antibodies of the present invention.Methods for modifying antibodies are already established in this field.

The antibodies of the present invention may also be bispecificantibodies. “Bispecific antibody” refers to an antibody that has in asingle molecule variable regions that recognize different epitopes. Theepitopes may be present in a single molecule or in separate molecules.

The antibody constant regions described above can be used as a constantregion in an antibody against an arbitrary antigen. The antigen is notparticularly limited.

The antibodies of the present invention can also be obtained by, forexample, the following methods. In one embodiment to obtain antibodiesof the present invention, one or more amino acid residues are firstdeleted or substituted with amino acids of interest in the constantregion. Methods for substituting one or more amino acid residues withamino acids of interest include, for example, site-directed mutagenesis(Hashimoto-Gotoh, T., Mizuno, T., Ogasahara, Y., and Nakagawa, M. Anoligodeoxyribonucleotide-directed dual amber method for site-directedmutagenesis. Gene (1995) 152, 271-275; Zoller, M. J., and Smith, M.Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors. Methods Enzymol. (1983) 100, 468-500; Kramer, W., Drutsa, V.,Jansen, H. W., Kramer, B., Pflugfelder, M., and Fritz, H. J. The gappedduplex DNA approach to oligonucleotide-directed mutation construction.Nucleic Acids Res. (1984) 12, 9441-9456; Kramer W., and Fritz H. J.Oligonucleotide-directed construction of mutations via gapped duplex DNAMethods. Enzymol. (1987) 154, 350-367; Kunkel, T. A. Rapid and efficientsite-specific mutagenesis without phenotypic selection. Proc. Natl.Acad. Sci. USA (1985) 82, 488-492). These methods can be used tosubstitute target amino acids in the constant region of an antibody withamino acids of interest.

In another embodiment to obtain antibodies, an antibody that binds to anantigen of interest is first prepared by methods known to those skilledin the art. When the prepared antibody is derived from a nonhumananimal, it can be humanized. The binding activity of the antibody can bedetermined by known methods. Next, one or more amino acid residues inthe constant region of the antibody are deleted or substituted withamino acids of interest.

More specifically, the present invention relates to methods forproducing antibodies, which comprise the steps of:

-   (a) expressing a DNA encoding an H chain in which one or more amino    acid residues in the constant region are deleted or substituted with    amino acids of interest, and a DNA encoding an L chain; and-   (b) collecting the expression products of step (a).

The first step of the production methods of the present invention isexpressing a DNA encoding an antibody H chain in which one or more aminoacid residues in the constant region are deleted or substituted withamino acids of interest, and a DNA encoding an antibody L chain. A DNAencoding an H chain in which one or more amino acid residues in theconstant region are deleted or substituted with amino acids of interestcan be prepared, for example, by obtaining a DNA encoding the constantregion of a wild type H chain, and introducing an appropriatesubstitution so that a codon encoding a particular amino acid in theconstant region encodes an amino acid of interest.

Alternatively, a DNA encoding an H chain in which one or more amino acidresidues in the constant region are deleted or substituted with aminoacids of interest can also be prepared by designing and then chemicallysynthesizing a DNA encoding a protein in which one or more amino acidresidues in the constant region of the wild type H chain are deleted orsubstituted with amino acids of interest.

The type of amino acid substitution includes the substitutions describedherein, but is not limited thereto.

Alternatively, a DNA encoding an H chain in which one or more amino acidresidues in the constant region are deleted or substituted with aminoacids of interest can also be prepared as a combination of partial DNAs.Such combinations of partial DNAs include, for example, the combinationof a DNA encoding a variable region and a DNA encoding a constantregion, and the combination of a DNA encoding an Fab region and a DNAencoding an Fc region, but are not limited thereto. A DNA encoding an Lchain can also be prepared as a combination of partial DNAs.

Methods for expressing the above-described DNAs include the methodsdescribed below. For example, an H chain expression vector isconstructed by inserting a DNA encoding an H chain variable region intoan expression vector along with a DNA encoding an H chain constantregion. Likewise, an L chain expression vector is constructed byinserting a DNA encoding an L chain variable region into an expressionvector along with a DNA encoding an L chain constant region.Alternatively, these H and L chain genes may be inserted into a singlevector. Expression vectors include, for example, SV40 virus-basedvectors, EB virus-based vectors, and BPV (papilloma virus)-basedvectors, but are not limited thereto.

Host cells are co-transformed with an antibody expression vectorconstructed by the methods described above. Such host cells include theabove-described cells such as CHO (Chinese hamster ovary) cells as wellas microorganisms such as E. coli, yeast, and Bacillus subtilis, andplants and animals (Nature Biotechnology (2007) 25, 563-565; NatureBiotechnology (1998) 16, 773-777; Biochemical and Biophysical ResearchCommunications (1999) 255, 444-450; Nature Biotechnology (2005) 23,1159-1169; Journal of Virology (2001) 75, 2803-2809; Biochemical andBiophysical Research Communications (2003) 308, 94-100). Thetransformation can be preferably achieved by using electroporation, thelipofectin method (R. W. Malone et al., Proc. Natl. Acad. Sci. USA(1989) 86, 6077; P. L. Felgner et al., Proc. Natl. Acad. Sci. USA (1987)84, 7413), calcium phosphate method (F. L. Graham & A. J. van der Eb,Virology (1973) 52, 456-467), DEAE-Dextran method, and the like.

In the next step of antibody production, the expression productsobtained in step (a) are collected. The expression products can becollected, for example, by culturing the transformants and thenseparating the products from the transformed cells or culture media.Separation and purification of antibodies can be achieved by anappropriate combination of methods such as centrifugation, ammoniumsulfate fractionation, salting out, ultrafiltration, columns of lq,FcRn, Protein A, and Protein G, affinity chromatography, ion exchangechromatography, and gel filtration chromatography.

<Methods for Improving the IgG2 Constant Region Stability under AcidicConditions>

The present invention also relates to methods for improving antibodystability under acidic conditions, which comprise the step ofsubstituting Met at position 276 (position 397 in the EU numberingsystem) in the amino acid sequence of SEQ ID NO: 2 (IgG2) with anotheramino acid. The methods of the present invention for improving antibodystability under acidic conditions may comprise other steps of amino acidsubstitution, as long as they comprise the step of substituting Met atposition 276 (position 397 in the EU numbering system) in the amino acidsequence of SEQ ID NO: 2 (IgG2) with another amino acid. The type ofamino acid after substitution is not particularly limited; however,substitution to Val is preferred. The method for amino acid substitutionis not particularly limited. The substitution can be achieved, forexample, by site-directed mutagenesis described above or a methoddescribed in the Examples.

<Methods for Reducing the Heterogeneity Originated from the Hinge Regionof IgG2 Constant Region>

The present invention also relates to methods for reducing antibodyheterogeneity, which comprise the step of substituting Cys at position14 (position 131 in the EU numbering system), Arg at position 16(position 133 in the EU numbering system), and/or Cys at position 102(position 219 in the EU numbering system) in the amino acid sequence ofSEQ ID NO: 2 (IgG2) with other amino acids. The type of amino acid aftersubstitution is not particularly limited; however, substitutions of Serfor Cys at position 14, Lys for Arg at position 16, and Ser for Cys atposition 102 are preferred. The methods of the present invention forreducing antibody heterogeneity may comprise other steps of amino acidsubstitution, as long as they comprise the step of substituting Cys atposition 14 (position 131 in the EU numbering system), Arg at position16 (position 133 in the EU numbering system), and/or Cys at position 102(position 219 in the EU numbering system) in the amino acid sequence ofSEQ ID NO: 2 (IgG2). The method for amino acid substitution is notparticularly limited. The substitutions can be achieved, for example, bysite-directed mutagenesis described above or a method described in theExamples. In the amino acid substitution, all of the three amino acidsdescribed above may be substituted or one or two (for example, positions14 and 102) of them may be substituted.

<Methods for Reducing the Heterogeneity Originated from Deletion ofC-terminal Amino Acids in an IgG2 Constant Region>

The present invention also relates to methods for reducing antibodyheterogeneity, which comprise the step of deleting Gly at position 325(position 446 in the EU numbering system) and Lys at position 326(position 447 in the EU numbering system) in an IgG2 constant regioncomprising the amino acid sequence of SEQ ID NO: 2. The methods of thepresent invention for reducing antibody heterogeneity may comprise othersteps of amino acid substitution, as long as they comprise the step ofdeleting Gly at position 325 (position 446 in the EU numbering system)and Lys at position 326 (position 447 in the EU numbering system) in anIgG2 constant region comprising the amino acid sequence of SEQ ID NO: 2.The method for amino acid substitution is not particularly limited. Thesubstitution can be achieved, for example, by site-directed mutagenesisdescribed above or a method described in the Examples.

<Methods for Improving the Pharmacokinetics by Substituting Amino Acidsof IgG2 Constant Region>

The present invention also relates to methods for improving thepharmacokinetics of an antibody, which comprise the step of substitutingHis at position 14 (EU268), Arg at position 234 (EU355), and/or Gln atposition 298 (EU419) in an IgG2 constant region comprising the aminoacid sequence of SEQ ID NO: 2. The methods of the present invention forimproving the pharmacokinetics of an antibody may comprise other stepsof amino acid substitution, as long as they comprise the above-describedstep. The type of amino acid after substitution is not particularlylimited; however, substitutions of Gln for His at position 147 (EU268),Gln for Arg at position 234 (EU355), and Glu for Gln at position 298(EU419) are preferred.

The present invention also relates to methods for improving thepharmacokinetics of an antibody, which comprise the step of substitutingAsn at position 313 (EU434) in an IgG2 constant region comprising theamino acid sequence of SEQ ID NO: 2 or 35 (M58). The type of amino acidafter substitution is not particularly limited; however, substitution toAla is preferred. The methods of the present invention for improving thepharmacokinetics of an antibody may comprise other steps of amino acidsubstitution, as long as they comprise the above-described step.

<Methods for Improving the Pharmacokinetics by Substituting Amino Acidsof IgG1 Constant Region>

The present invention also relates to methods for improving thepharmacokinetics of an antibody, which comprise the step of substitutingAsn at position 317 (EU434) in an IgG1 constant region comprising theamino acid sequence of SEQ ID NO: 1. The type of amino acid aftersubstitution is not particularly limited; however, substitution to Alais preferred. The methods of the present invention for improving thepharmacokinetics of an antibody may comprise other steps of amino acidsubstitution, as long as they comprise the above-described step.

The present invention also relates to methods for improving thepharmacokinetics of an antibody and reducing the heterogeneityoriginated from deletion of C-terminal amino acids, which comprise thestep of substituting Asn at position 317 (EU434) and deleting Gly atposition 329 (EU446) and Lys at position 330 (EU447) in an IgG1 constantregion comprising the amino acid sequence of SEQ ID NO: 1. The type ofamino acid after substitution is not particularly limited; however,substitution to Ala is preferred. The methods of the present inventionfor improving the pharmacokinetics of an antibody may comprise othersteps of amino acid substitution, as long as they comprise theabove-described step.

<Methods for Reducing the FcγR Binding while Maintaining the HumanSequence in the IgG2 Constant Region>

The present invention also relates to methods for reducing the FcγRbinding of an antibody, which comprise the step of substituting Ser forAla at position 209 (EU330), Ser for Pro at position 210 (EU331), andAla for Thr at position 218 (EU339) in an IgG2 constant regioncomprising the amino acid sequence of SEQ ID NO: 2. The methods of thepresent invention for reducing the FcγR binding of an antibody maycomprise other steps of amino acid substitution, as long as theycomprise the step of substituting Ser for Ala at position 209 (EU330),Ser for Pro at position 210 (EU331), and Ala for Thr at position 218(EU339) in an IgG2 constant region comprising the amino acid sequence ofSEQ ID NO: 2. The method for amino acid substitution is not particularlylimited. The substitution can be achieved, for example, by site-directedmutagenesis described above or a method described in the Examples.

The present invention also relates to methods for reducing theheterogeneity originated from the hinge region of IgG2, methods forimproving antibody stability under acidic conditions, methods forreducing antibody heterogeneity originated from C-terminus, and/ormethods for reducing the FcγR binding of an antibody, all of whichcomprise, in an IgG2 constant region comprising the amino acid sequenceof SEQ ID NO: 2 (M14ΔGK), the steps of:

-   (a) substituting Ala at position 209 (position 330 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (b) substituting Pro at position 210 (position 331 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (c) substituting Thr at position 218 (position 339 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (d) substituting Met at position 276 (position 397 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (e) substituting Cys at position 14 (position 131 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (f) substituting Arg at position 16 (position 133 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (g) substituting Cys at position 102 (position 219 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (h) substituting Glu at position 20 (position 137 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (i) substituting Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid; and-   (j) deleting Gly at position 325 and Lys at position 326 (positions    446 and 447 in the EU numbering system, respectively) in the amino    acid sequence of SEQ ID NO: 2.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Ala at position 209 (position 330 inthe EU numbering system), Ser for Pro at position 210 (position 331 inthe EU numbering system), Ala for Thr at position 218 (position 339 inthe EU numbering system), Val for Met at position 276 (position 397 inthe EU numbering system), Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Ser for Cys at position 102 (position 219 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), and Gly for Ser at position 21 (position 138in the EU numbering system) are preferred.

The present invention also relates to methods for reducing theheterogeneity originated from the hinge region of IgG2, methods forreducing antibody heterogeneity originated from C-terminus, and/ormethods for reducing the FcγR binding of an antibody, all of whichcomprise, in an IgG2 constant region comprising the amino acid sequenceof SEQ ID NO: 2 (M86ΔGK), the steps of:

-   (a) substituting Ala at position 209 (position 330 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (b) substituting Pro at position 210 (position 331 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (c) substituting Thr at position 218 (position 339 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (d) substituting Cys at position 14 (position 131 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (e) substituting Arg at position 16 (position 133 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (f) substituting Cys at position 102 (position 219 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (g) substituting Glu at position 20 (position 137 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (h) substituting Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid; and-   (i) deleting Gly at position 325 and Lys at position 326 (positions    446 and 447 in the EU numbering system, respectively) in the amino    acid sequence of SEQ ID NO: 2.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Ala at position 209 (position 330 inthe EU numbering system), Ser for Pro at position 210 (position 331 inthe EU numbering system), Ala for Thr at position 218 (position 339 inthe EU numbering system), Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Ser for Cys at position 102 (position 219 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), and Gly for Ser at position 21 (position 138in the EU numbering system) are preferred.

The methods of the present invention may comprise other steps such asamino acid substitution and deletion, as long as they comprise the stepsdescribed above. The methods for amino acid substitution and deletionare not particularly limited. The substitution and deletion can beachieved, for example, by site-directed mutagenesis described above or amethod described in the Examples.

The present invention also relates to methods for reducing theheterogeneity originated from the hinge region of IgG2, methods forimproving antibody stability under acidic conditions, and/or methods forreducing antibody heterogeneity originated from C-terminus, all of whichcomprise in an IgG2 constant region comprising the amino acid sequenceof SEQ ID NO: 2 (M31ΔGK), the steps of:

-   (a) substituting Met at position 276 (position 397 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (b) substituting Cys at position 14 (position 131 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (c) substituting Arg at position 16 (position 133 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (d) substituting Cys at position 102 (position 219 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (e) substituting Glu at position 20 (position 137 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (f) substituting Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid; and-   (g) deleting Gly at position 325 and Lys at position 326 (positions    446 and 447 in the EU numbering system, respectively) in the amino    acid sequence of SEQ ID NO: 2.

The type of amino acid after substitution is not particularly limited;however, substitutions of Val for Met at position 276 (position 397 inthe EU numbering system), Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Ser for Cys at position 102 (position 219 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), and Gly for Ser at position 21 (position 138in the EU numbering system) are preferred.

The present invention further relates to methods for reducing theheterogeneity originated from the hinge region of IgG2 and/or methodsfor reducing antibody heterogeneity originated from C-terminus, all ofwhich comprise in an IgG2 constant region comprising the amino acidsequence of SEQ ID NO: 2 (M40ΔGK), the steps of:

-   (a) substituting Cys at position 14 (position 131 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (b) substituting Arg at position 16 (position 133 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (c) substituting Cys at position 102 (position 219 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (d) substituting Glu at position 20 (position 137 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid;-   (e) substituting Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2 with    another amino acid; and-   (f) deleting Gly at position 325 and Lys at position 326 (positions    446 and 447 in the EU numbering system, respectively) in the amino    acid sequence of SEQ ID NO: 2.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Ser for Cys at position 102 (position 219 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), and Gly for Ser at position 21 (position 138in the EU numbering system) are preferred.

The present invention also relates to methods for reducing antibodyheterogeneity originated from the hinge region of IgG2, methods forimproving pharmacokinetics, and/or methods for reducing antibodyheterogeneity originated from C-terminus, all of which comprise in anIgG2 constant region comprising the amino acid sequence of SEQ ID NO: 2(M58), the steps of:

-   (a) substituting Ser for Cys at position 14 (position 131 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (b) substituting Lys for Arg at position 16 (position 133 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (c) substituting Ser for Cys at position 102 (position 219 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (d) substituting Gly for Glu at position 20 (position 137 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (e) substituting Gly for Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (f) substituting Gln for His at position 147 (position 268 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (g) substituting Gln for Arg at position 234 (position 355 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (h) substituting Glu for Gln at position 298 (position 419 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2; and-   (i) deleting Gly at position 325 and Lys at position 326 (positions    446 and 447 in the EU numbering system, respectively) in the amino    acid sequence of SEQ ID NO: 2.

The present invention also relates to methods for reducing antibodyheterogeneity originated from the hinge region of IgG2, methods forimproving pharmacokinetics, and/or methods for reducing antibodyheterogeneity originated from C-terminus, all of which comprise in anIgG2 constant region comprising the amino acid sequence of SEQ ID NO: 2(M73), the steps of:

-   (a) substituting Ser for Cys at position 14 (position 131 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (b) substituting Lys for Arg at position 16 (position 133 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (c) substituting Ser for Cys at position 102 (position 219 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (d) substituting Gly for Glu at position 20 (position 137 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (e) substituting Gly for Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (f) substituting Gln for His at position 147 (position 268 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (g) substituting Gln for Arg at position 234 (position 355 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (h) substituting Glu for Gln at position 298 (position 419 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2;-   (i) substituting Ala for Asn at position 313 (position 434 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 2; and-   (j) deleting Gly at position 325 and Lys at position 326 (positions    446 and 447 in the EU numbering system, respectively) in the amino    acid sequence of SEQ ID NO: 2.

The methods of the present invention may comprise other steps such asamino acid substitution and deletion, as long as they comprise the stepsdescribed above. The methods for amino acid substitution and deletionare not particularly limited. The substitution and deletion can beachieved, for example, by site-directed mutagenesis described above or amethod described in the Examples.

<Methods for Improving the Stability of an IgG4 Constant Region underAcidic Conditions>

The present invention also relates to methods for improving antibodystability under acidic conditions, which comprise the step ofsubstituting Arg at position 289 (position 409 in the EU numberingsystem) of an IgG4 constant region comprising the amino acid sequence ofSEQ ID NO: 3 with another amino acid. The methods of the presentinvention for improving antibody stability under acidic conditions maycomprise other steps of amino acid substitution, as long as theycomprise the step of substituting Arg at position 289 (position 409 inthe EU numbering system) in the amino acid sequence of SEQ ID NO: 3(human IgG4 constant region) with another amino acid. The type of aminoacid after substitution is not particularly limited; however,substitution to Lys is preferred. The method for amino acid substitutionis not particularly limited. The substitution can be achieved, forexample, by site-directed mutagenesis described above or a methoddescribed in the Examples.

<Methods for Reducing the Heterogeneity Originated from Deletion ofC-terminal Amino Acids in an IgG4 Constant Region>

The present invention also relates to methods for reducing theheterogeneity of an antibody, which comprise the step of deleting Gly atposition 326 (position 446 in the EU numbering system) and Lys atposition 327 (position 447 in the EU numbering system) in an IgG4constant region comprising the amino acid sequence of SEQ ID NO: 3 (Mol.Immunol. 1993 January; 30(1):105-8). The methods of the presentinvention for reducing the heterogeneity may comprise other steps ofamino acid substitution, as long as they comprise the step of deletingLys at position 327 (position 447 in the EU numbering system) and/or Glyat position 326 (position 446 in the EU numbering system) in an IgG4constant region comprising the amino acid sequence of SEQ ID NO: 3. Themethod for amino acid substitution is not particularly limited. Thesubstitution can be achieved, for example, by site-directed mutagenesisdescribed above or a method described in the Examples.

The present invention also relates to methods for improving thestability under acidic conditions, methods for reducing theheterogeneity originated from C-terminus, and/or methods for reducingthe FcγR binding of an antibody, all of which comprise, in an IgG4constant region comprising the amino acid sequence of SEQ ID NO: 3(M11ΔGK), the steps of:

-   (a) substituting Cys at position 14 (position 131 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (b) substituting Arg at position 16 (position 133 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (c) substituting Glu at position 20 (position 137 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (d) substituting Ser at position 21 (position 138 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (e) substituting Arg at position 97 (position 214 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (f) substituting Ser at position 100 (position 217 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (g) substituting Tyr at position 102 (position 219 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (h) substituting Gly at position 103 (position 220 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (i) substituting Pro at position 104 (position 221 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (j) substituting Pro at position 105 (position 222 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (k) substituting Glu at position 113 (position 233 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (l) substituting Phe at position 114 (position 234 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (m) substituting Leu at position 115 (position 235 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid;-   (n) deleting Gly at position 116 (position 236 in the EU numbering    system) in the amino acid sequence of SEQ ID NO: 3;-   (o) substituting Arg at position 289 (position 409 in the EU    numbering system) in the amino acid sequence of SEQ ID NO: 3 with    another amino acid; and-   (p) deleting Gly at position 236 and Lys at position 237 (positions    446 and 447 in the EU numbering system, respectively) in the amino    acid sequence of SEQ ID NO: 3.

The type of amino acid after substitution is not particularly limited;however, substitutions of Ser for Cys at position 14 (position 131 inthe EU numbering system), Lys for Arg at position 16 (position 133 inthe EU numbering system), Gly for Glu at position 20 (position 137 inthe EU numbering system), Gly for Ser at position 21 (position 138 inthe EU numbering system), Thr for Arg at position 97 (position 214 inthe EU numbering system), Arg for Ser at position 100 (position 217 inthe EU numbering system), Ser for Tyr at position 102 (position 219 inthe EU numbering system), Cys for Gly at position 103 (position 220 inthe EU numbering system), Val for Pro at position 104 (position 221 inthe EU numbering system), Glu for Pro at position 105 (position 222 inthe EU numbering system), Pro for Glu at position 113 (position 233 inthe EU numbering system), Val for Phe at position 114 (position 234 inthe EU numbering system), Ala for Leu at position 115 (position 235 inthe EU numbering system), and Lys for Arg at position 289 (position 409in the EU numbering system) are preferred.

The methods of the present invention may comprise other steps, such asamino acid substitution and deletion, as long as they comprise the stepsdescribed above. The method for amino acid substitution and deletion arenot particularly limited. The substitution and deletion can be achieved,for example, by site-directed mutagenesis described above or a methoddescribed in the Examples.

<Methods for Reducing the Heterogeneity Originated from Deletion ofC-terminal Amino Acids in an IgG1 Constant Region>

The present invention also relates to methods for reducing antibodyheterogeneity, which comprise the step of deleting Gly at position 329(position 446 in the EU numbering system) and Lys at position 330(position 447 in the EU numbering system) in an IgG1 constant regioncomprising the amino acid sequence of SEQ ID NO: 1. The methods of thepresent invention for reducing antibody heterogeneity may comprise othersteps of amino acid substitutions, as long as they comprise the step ofdeleting Lys at position 330 (position 447 in the EU numbering system)and Gly at position 329 (position 446 in the EU numbering system) in anIgG1 constant region comprising the amino acid sequence of SEQ ID NO: 1.The method for amino acid substitution is not particularly limited. Thesubstitution can be achieved, for example, by site-directed mutagenesisdescribed above or a method described in the Examples.

The antibody constant regions described above are not particularlylimited, and may be used for any antibodies. Examples of antibodieswhich use the constant region of the present invention include:

-   (a) a heavy chain that comprises the amino acid sequence of SEQ ID    NO: 48 (VH4-M73);-   (b) a heavy chain that comprises the amino acid sequence of SEQ ID    NO: 46 (VH3-M73);-   (c) a heavy chain that comprises the amino acid sequence of SEQ ID    NO: 44 (VH5-M83);-   (d) a light chain that comprises the amino acid sequence of SEQ ID    NO: 49 (VL1-kappa);-   (e) a light chain that comprises the amino acid sequence of SEQ ID    NO: 47 (VL3-kappa);-   (f) a light chain that comprises the amino acid sequence of SEQ ID    NO: 45 (VL5-kappa);-   (g) an antibody that comprises the heavy chain of (a) and the light    chain of (d) (FV3-M73);-   (h) an antibody that comprises the heavy chain of (b) and the light    chain of (e) (FV4-M73); and-   (i) an antibody that comprises the heavy chain of (c) and the light    chain of (f) (FV5-M83).    <Pharmaceutical Compositions Comprising Antibodies>

The present invention provides pharmaceutical compositions comprising anantibody of the present invention.

The pharmaceutical compositions of the present invention can beformulated, in addition to the antibodies, with pharmaceuticallyacceptable carriers by known methods. For example, the compositions canbe used parenterally, when the antibodies are formulated in a sterilesolution or suspension for injection using water or any otherpharmaceutically acceptable liquid. For example, the compositions can beformulated by appropriately combining the antibodies withpharmaceutically acceptable carriers or media, specifically, sterilewater or physiological saline, vegetable oils, emulsifiers, suspendingagents, surfactants, stabilizers, flavoring agents, excipients,vehicles, preservatives, binding agents, and such, by mixing them at aunit dose and form required by generally accepted pharmaceuticalimplementations. The content of the active ingredient in such aformulation is adjusted so that an appropriate dose within the requiredrange can be obtained.

Sterile compositions for injection can be formulated using vehicles suchas distilled water for injection, according to standard protocols.

Aqueous solutions used for injection include, for example, physiologicalsaline and isotonic solutions containing glucose or other adjuvants suchas D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These can beused in conjunction with suitable solubilizers such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, and non-ionic surfactants such as Polysorbate 80™and HCO-50.

Oils include sesame oils and soybean oils, and can be combined withsolubilizers such as benzyl benzoate or benzyl alcohol. These may alsobe formulated with buffers, for example, phosphate buffers or sodiumacetate buffers; analgesics, for example, procaine hydrochloride;stabilizers, for example, benzyl alcohol or phenol; or antioxidants. Theprepared injections are typically aliquoted into appropriate ampules.

The administration is preferably carried out parenterally, andspecifically includes injection, intranasal administration,intrapulmonary administration, and percutaneous administration. Forexample, injections can be administered systemically or locally byintravenous injection, intramuscular injection, intraperitonealinjection, or subcutaneous injection.

Furthermore, the method of administration can be appropriately selectedaccording to the age and symptoms of the patient. A single dose of thepharmaceutical composition containing an antibody or a polynucleotideencoding an antibody can be selected, for example, from the range of0.0001 to 1,000 mg per kg of body weight. Alternatively, the dose maybe, for example, in the range of 0.001 to 100,000 mg/person. However,the dose is not limited to these values. The dose and method ofadministration vary depending on the patient's body weight, age, andsymptoms, and can be appropriately selected by those skilled in the art.

As used herein, the three-letter and single-letter codes for respectiveamino acids are as follows:

-   Alanine: Ala (A)-   Arginine: Arg (R)-   Asparagine: Asn (N)-   Aspartic acid: Asp (D)-   Cysteine: Cys (C)-   Glutamine: Gln (Q)-   Glutamic acid: Glu (E)-   Glycine: Gly (G)-   Histidine: His (H)-   Isoleucine: Ile (I)-   Leucine: Leu (L)-   Lysine: Lys (K)-   Methionine: Met (M)-   Phenylalanine: Phe (F)-   Proline: Pro (P)-   Serine: Ser (S)-   Threonine: Thr (T)-   Tryptophan: Trp (W)-   Tyrosine: Tyr (Y)-   Valine: Val (V)

All prior art documents cited herein are incorporated by reference intheir entirety.

EXAMPLES

Hereinbelow, the present invention is further specifically describedwith reference to the Examples, but it is not to be construed as beinglimited thereto.

Example 1 Improvement of the Stability of IgG2 and IgG4 Under AcidicCondition

Construction of Expression Vectors for IgG2- or IgG4-converted HumanizedIL-6 Receptor Antibodies and Expression of the Antibodies

To reduce the Fcγ receptor-binding activity, the constant region of ahumanized anti-human IL-6 receptor antibody, humanized PM-1 antibody(Cancer Res. 1993 Feb. 15; 53(4):851-6), which is of the IgG1 isotype,was substituted with IgG2 or IgG4 (Mol. Immunol. 1993 January;30(1):105-8) to generate molecules WT-IgG2 (SEQ ID NO: 13) and WT-IgG4(SEQ ID NO: 14). An animal cell expression vector was used to expressthe IgGs. An expression vector, in which the constant region ofhumanized PM-1 antibody (IgG1) used in Reference Example 1 was digestedwith NheI/NotI and then substituted with the IgG2 or IgG4 constantregion by ligation, was constructed. The nucleotide sequence of each DNAfragment was determined with a DNA sequencer (ABI PRISM 3730xL DNASequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems)) usingthe BigDye Terminator Cycle Sequencing Kit (Applied Biosystems)according to the attached instruction manual. Using the WT L chain (SEQID NO: 15), WT-IgG1, WT-IgG2, and WT-IgG4 were expressed by the methoddescribed below. Human embryonic kidney cancer-derived HEK293H cells(Invitrogen) were suspended in DMEM (Invitrogen) supplemented with 10%Fetal Bovine Serum (Invitrogen). The cells (10-ml/plate; cell density of5 to 6×10⁵ cells/ml) were plated on dishes for adherent cells (10 cm indiameter; CORNING) and cultured in a CO₂ incubator (37° C., 5% CO₂) forone whole day and night. Then, the medium was removed by aspiration, and6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added. The preparedplasmid DNA mixture (13.8 μg in total) was combined with 20.7 μl of 1μg/ml Polyethylenimine (Polysciences Inc.) and 690 μl of CHO-S-SFMIImedium. The resulting mixture was incubated at room temperature for 10minutes, and then added to the cells in each dish. The cells wereincubated in a CO₂ incubator (at 37° C. under 5% CO₂) for 4 to 5 hours.Then, 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added to thedishes, and the cells were incubated in a CO₂ incubator for three days.The culture supernatants were collected and centrifuged (approx. 2000 g,5 min, room temperature) to remove the cells, and sterilized through0.22-μm filter MILLEX®-GV (Millipore). The samples were stored at 4° C.until use.

(1) Humanized PM-1 antibody (PM-1 VH+IgG1) H chain, SEQ ID NO: 12 (aminoacid sequence)

(2) Humanized PM-1 VH+IgG2 H chain, SEQ ID NO: 13 (amino acid sequence)

(3) Humanized PM-1 VH+IgG4 H chain, SEQ ID NO: 14 (amino acid sequence)

Purification of WT-IgG1, WT-IgG2, and WT-IgG4 through Elution fromProtein A using Hydrochloric Acid

50 μl of rProtein A Sepharose™ Fast Flow (Amersham Biosciences)suspended in TBS was added to the obtained culture supernatants, and thecombined solutions were mixed by inversion at 4° C. for four hours ormore. The solutions were transferred into 0.22-μm filter cups ofUltrafree®-MC (Millipore). After washing three times with 500 μl of TBS,the rProtein A Sepharose™ resins were suspended in 100 μl of 10 mMHCl/150 mM NaCl (pH 2.0) and the mixtures were incubated for two minutesto elute the antibodies (hydrochloric acid elution). Immediately, theeluates were neutralized by adding 6.7 μl of 1.5 M Tris-HCl (pH 7.8).The elution was carried out twice, yielding 200 μl of purifiedantibodies.

Gel Filtration Chromatography Analysis of WT-IgG1, WT-IgG2, and WT-IgG4Purified by Hydrochloric Acid Elution

The contents of aggregate in the purified samples obtained byhydrochloric acid elution were assessed by gel filtration chromatographyanalysis.

Aggregation Assessment Method:

-   -   System: Waters Alliance    -   Column: G3000SWxl (TOSOH)    -   Mobile phase: 50 mM sodium phosphate, 300 mM KCl pH 7.0    -   Flow rate, wavelength: 0.5 ml/min, 220 nm

The result is shown in FIG. 1. While the content of aggregate in WT-IgG1after purification was about 2%, those of WT-IgG2 and WT-IgG4 afterpurification were about 25%. This suggests that IgG1 is stable to acidduring hydrochloric acid elution, and by contrast, IgG2 and IgG4 areunstable and underwent denaturation/aggregation. Thus, the stability ofIgG2 and IgG4 under acidic condition was demonstrated to be lower thanthat of IgG1. Protein A has been frequently used to purify IgGmolecules, and the IgG molecules are eluted from Protein A under acidiccondition. In addition, virus inactivation, which is required whendeveloping IgG molecules as pharmaceuticals, is generally carried outunder acidic condition. It is thus desirable that the stability of IgGmolecules under acidic condition is higher. However, the stability ofIgG2 and IgG4 molecules under acidic condition was found to be lowerthan that of IgG1, and suggests for the first time that there is aproblem of denaturation/aggregation under acidic condition in developingIgG2 and IgG4 molecules as pharmaceuticals. It is desirable that thisproblem of denaturation/aggregation be overcome when developing them aspharmaceuticals. To date, however, no report has been published on amethod for solving this problem through amino acid substitution.

Preparation and Assessment of WT-IgG2 and WT-IgG4 having an Altered CH3Domain

The stability of IgG2 and IgG4 molecules under acidic condition wasdemonstrated to be lower than that of IgG1. Thus, altered forms of IgG2and IgG4 molecules were tested to improve the stability under acidiccondition. According to models for the constant regions of IgG2 and IgG4molecules, one of the potential destabilizing factors under acidiccondition was thought to be the instability at the CH3-CH3 domaininterface. Methionine at position 397 in the EU numbering system inIgG2, or arginine at position 409 in the EU numbering system in IgG4 wasthought to destabilize the CH3/CH3 interface. Since positions 397 and409 of IgG1 in the EU numbering system are valine and lysine,respectively, an altered IgG2 antibody that comprises the substitutionof valine for methionine at position 397 in the EU numbering system(IgG2-M397V, SEQ ID NO: 16 (amino acid sequence)) and an altered IgG4antibody that comprises the substitution of lysine for arginine atposition 409 in the EU numbering system (IgG4-R409K, SEQ ID NO: 17(amino acid sequence)) are prepared.

The methods used for constructing expression vectors for the antibodiesof interest, and expressing and purifying the antibodies, were the sameas those used for the hydrochloric acid elution described above. Gelfiltration chromatography analysis was carried out to estimate thecontents of aggregate in the purified samples obtained by hydrochloricacid elution from Protein A.

Aggregation Assessment Method:

-   -   System: Waters Alliance    -   Column: G3000SWxl (TOSOH)    -   Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0    -   Flow rate, wavelength: 0.5 ml/min, 220 nm

The result is shown in FIG. 1. While the content of aggregate in WT-IgG1after purification was about 2%, those in WT-IgG2 and WT-IgG4 afterpurification were about 25%. By contrast, the contents of aggregate invariants with altered CH3 domain, IgG2-M397V and IgG4-R409K, werecomparable (approx. 2%) to that in IgG1. This finding demonstrates thatthe stability of an IgG2 or IgG4 antibody under acidic condition can beimproved by substituting valine for methionine of IgG2 at position 397in the EU numbering system or lysine for arginine of IgG4 at position409 in the EU numbering system, respectively. The purified antibodies ofwere dialyzed against a solution of 20 mM sodium acetate, 150 mM NaCl,pH 6.0 (EasySEP, TOMY). DSC measurement (measurements of midpointtemperature and Tm value) was carried out at a heating rate of 1° C./minfrom 40 to 100° C. at a protein concentration of about 0.1 mg/ml.Furthermore, the midpoint temperatures of thermal denaturation ofWT-IgG2, WT-IgG4, IgG2-M397V, and IgG4-R409K were determined. The resultshowed that the Tm value for the altered CH3 domain was higher inIgG2-M397V and IgG4-R409K as compared to WT-IgG2 and WT-IgG4,respectively. This suggests that IgG2-M397V and IgG4-R409K are alsosuperior in terms of thermal stability as compared to WT-IgG2 andWT-IgG4, respectively.

IgG2 and IgG4 are exposed to acidic condition in virus inactivationprocess and in the purification process using Protein A. Thus,denaturation/aggregation in the above processes was problematic.However, it was discovered that the problem could be solved by usingIgG2-M397V and IgG4-R409K for the sequences of IgG2 and IgG4 constantregions. Thus, these alterations were revealed to be very useful indeveloping IgG2 and IgG4 antibody pharmaceuticals. Furthermore, theusefulness of IgG2-M397V and IgG4-R409K was also demonstrated by thefinding that they are superior in thermal stability.

Example 2 Improvement of Heterogeneity Derived from Disulfide Bonds inIgG2

Purification of WT-IgG1, WT-IgG2, and WT-IgG4 through Acetic AcidElution from Protein A

50 μl of rProtein A Sepharose™ Fast Flow (Amersham Biosciences)suspended in TBS was added to the culture supernatants obtained inExample 1, and the combined solutions were mixed by inversion at 4° C.for four hours or more. The solutions were transferred into 0.22-μmfilter cups of Ultrafree®-MC (Millipore). After washing three times with500 μl of TBS, the rProtein A Sepharose™ resins were suspended in 100 μlof aqueous solution of 50 mM sodium acetate (pH 3.3) and the mixtureswere incubated for two minutes to elute the antibodies. Immediately, theeluates were neutralized by adding 6.7 μl of 1.5 M Tris-HCl (pH 7.8).The elution was carried out twice, yielding 200 μl of purifiedantibodies.

Analysis of WT-IgG1, WT-IgG2, and WT-IgG4 by Cation ExchangeChromatography (IEC)

Purified WT-IgG1, WT-IgG2, and WT-IgG4 were analyzed for homogeneity bycation exchange chromatography.

Assessment Method using IEC:

-   -   System: Waters Alliance    -   Column: ProPac WCX-10 (Dionex)    -   Mobile phase A: 25 mM MES-NaOH, pH 6.1        -   B: 25 mM MES-NaOH, 250 mM Na-Acetate, pH 6.1    -   Flow rate, wavelength: 0.5 ml/min, 280 nm    -   GradientB: 50%-75% (75 min) in the analysis of WT-IgG1        -   B: 30%-55% (75 min) in the analysis of WT-IgG2 and WT-IgG4

The result is shown in FIG. 2. WT-IgG2 showed more than one peak in theion exchange analysis while WT-IgG1 and WT-IgG4 exhibited a single peak.This suggests that the IgG2 molecule is more heterogeneous as comparedto IgG1 and IgG4. Indeed, IgG2 isotypes have been reported to haveheterogeneity derived from disulfide bonds in the hinge region(Non-patent Document 10). Thus, the hetero-peaks of IgG2 shown in FIG. 2are also assumed to be objective substance/related substances derivedfrom the disulfide bonds. It is not easy to manufacture them as apharmaceutical in large-scale while maintaining the objectivesubstances/related substances related heterogeneity between productions.Thus, homogeneous (less heterogeneous) substances are desirable as muchas possible for antibody molecules developed as pharmaceuticals. Forwild type IgG2, there is a problem of homogeneity which is important indeveloping antibody pharmaceuticals. Indeed, US20060194280 (A1) hasshown that natural IgG2 gives various hetero-peaks as a result of thedisulfide bonds in ion exchange chromatography analysis, and that thebiological activity varies among these peaks. US20060194280 (A1) reportsrefolding in the purification process as a method for combining thehetero-peaks into a single one, but use of such a process in theproduction is costly and complicated. Thus, a preferred method forcombining the hetero-peaks into a single one is based on amino acidsubstitution. Although the heterogeneity originated from disulfide bondsin the hinge region should be overcome to develop IgG2 aspharmaceuticals, no report has been published to date on a method forsolving this problem through amino acid substitution.

Preparation and Assessment of Altered WT-IgG2 CH1 Domain and HingeRegion

As shown in FIG. 3, there are various potential disulfide bond patternsfor an IgG2 molecule. Possible causes of the heterogeneity derived fromthe hinge region of IgG2 were differential pattern of disulfide bondingand free cysteines. IgG2 has two cysteines (at positions 219 and 220 inthe EU numbering system) in the upper hinge region, and cysteinesadjacent to the two upper-hinge cysteines include cysteine at position131 in the EU numbering system in the H chain CH1 domain and L chainC-terminal cysteine, and two corresponding cysteines in the H chainupper hinge of the dimerization partner. Specifically, there are eightcysteines in total in the vicinity of the upper hinge region of IgG2when the antibody is in the associated form of H2L2. This may be thereason for the various heterogeneous patterns due to wrong disulfidebonding and free cysteines.

The hinge region sequence and CH1 domain of IgG2 were altered to reducethe heterogeneity originated from the IgG2 hinge region. Examinationswere conducted to avoid the heterogeneity of IgG2 due to differentialpattern of disulfide bonding and free cysteines. The result of examiningvarious altered antibodies suggested that the heterogeneity could beavoided without decreasing the thermal stability by substituting serineand lysine for cysteine and arginine at positions 131 and 133 in the EUnumbering system, respectively, in the H chain CH1 domain, andsubstituting serine for cysteine at position 219, EU numbering, in theupper hinge of H chain of the wild type IgG2 constant region sequence(hereinafter IgG2-SKSC) (IgG2-SKSC, SEQ ID NO: 18). These substitutionswould enable IgG2-SKSC to form a homogenous covalent bond between H andL chains, which is a disulfide bond between the C-terminal cysteine ofthe L chain and cysteine at position 220 in the EU numbering system(FIG. 4).

The methods described in Reference Example 1 were used to construct anexpression vector for IgG2-SKSC and to express and purify IgG2-SKSC. Thepurified IgG2-SKSC and wild type IgG2 (WT-IgG2) were analyzed forhomogeneity by cation exchange chromatography.

Assessment Method using IEC:

-   -   System: Waters Alliance    -   Column: ProPac WCX-10 (Dionex)    -   Mobile phase A: 25 mM MES-NaOH, pH 5.6        -   B: 25 mM MES-NaOH, 250 mM Na-Acetate, pH 5.6    -   Flow rate, wavelength: 0.5 ml/min, 280 nm    -   Gradient B: 50%-100% (75 min)

The result is shown in FIG. 5. As expected above, IgG2-SKSC was shown tobe eluted at a single peak while WT-IgG2 gave multiple peaks. Thissuggests that the heterogeneity derived from disulfide bonds in thehinge region of IgG2 can be avoided by using alterations such as thoseused to generate IgG2-SKSC, which allow formation of a single disulfidebond between the C-terminal cysteine of the L chain and cysteine atposition 220 in the EU numbering system. The midpoint temperatures ofthermal denaturation of WT-IgG1, WT-IgG2, and IgG2-SKSC were determinedby the same methods as described in Example 1. The result showed thatWT-IgG2 gave a peak for Fab domain which has a lower Tm value thanWT-IgG1, while IgG2-SKSC did not give such a peak. This suggests thatIgG2-SKSC is also superior in thermal stability as compared to WT-IgG2.

Although wild type IgG2 was thought to have a homogeneity problem whichis important in developing antibody pharmaceuticals, it was found thatthis problem could be solved by using IgG2-SKSC for the constant regionsequence of IgG2. Thus, IgG2-SKSC is very useful in developing IgG2antibody pharmaceuticals. Furthermore, the usefulness of IgG2-SKSC wasalso demonstrated by the finding that it is superior in thermalstability.

Example 3 Improvement of C-terminal Heterogeneity in IgG Molecules

Construction of an Expression Vector for H Chain C-terminal ΔGK Antibodyfrom WT-IgG1

For heterogeneity of the C-terminal sequences of an antibody, deletionof C-terminal amino acid lysine residue, and amidation of the C-terminalamino group due to deletion of both of the two C-terminal amino acids,glycine and lysine, have been reported (Non-patent Document 12). Theabsence of such heterogeneity is preferred when developing antibodypharmaceuticals. Actually, in humanized PM-1 antibody TOCILIZUMAB, themajor component is the sequence that lacks the C-terminal amino acidlysine, which is encoded by the nucleotide sequence but deleted inpost-translational modification, and the minor component having thelysine also coexists as heterogeneity. Thus, the C-terminal amino acidsequence was altered to reduce the C-terminal heterogeneity.Specifically, the present inventors altered the nucleotide sequence ofwild type IgG1 to delete the C-terminal lysine and glycine from the Hchain constant region of the IgG1, and assessed whether the amidation ofthe C-terminal amino group could be suppressed by deleting the twoC-terminal amino acids glycine and lysine.

Mutations were introduced into the C-terminal sequence of the H chainusing pB-CH vector encoding the humanized PM-1 antibody (WT) obtained inReference Example 1. The nucleotide sequence encoding Lys at position447 and/or Gly at position 446 in the EU numbering system was convertedinto a stop codon by introducing a mutation using the QuikChangeSite-Directed Mutagenesis Kit (Stratagene) according to the methoddescribed in the attached instruction manual. Thus, expression vectorsfor antibody engineered to lack the C-terminal amino acid lysine(position 447 in the EU numbering system) and antibody engineered tolack the two C-terminal amino acids glycine and lysine (positions 446and 447 in the EU numbering system, respectively) were constructed. Hchain C-terminal ΔK and ΔGK antibodies were obtained by expressing theengineered H chains and the L chain of the humanized PM-1 antibody. Theantibodies were expressed and purified by the method described inReference Example 1.

Purified H chain C-terminal ΔGK antibody was analyzed by cation exchangechromatography according to the following procedure. The effect of theC-terminal deletion on heterogeneity was assessed by cation exchangechromatography analysis using the purified H chain C-terminal ΔGKantibody according to the method described below. The conditions ofcation exchange chromatography analysis are described below.Chromatograms for humanized PM-1 antibody, H chain C-terminal ΔKantibody, and H chain C-terminal ΔGK antibody were compared.

-   -   Column: ProPac WCX-10 (Dionex)    -   Mobile phase A: 25 mmol/l MES/NaOH, pH 6.1        -   B: 25 mmol/l MES/NaOH, 250 mmol/l NaCl, pH 6.1    -   Flow rate: 0.5 ml/min    -   Gradient: 25% B (5 min)->(105 min)->67% B->(1 min)->100% B (5        min)    -   Detection: 280 nm

The analysis result for the non-altered humanized PM-1 antibody, H chainC-terminal ΔK antibody, and H chain C-terminal ΔGK antibody is shown inFIG. 6. Non-patent Document 10 reports a basic peak with more prolongedretention time than that of the main peak. Analysis of a basic peakcorresponding to that reported in Non-patent Document 10 shows that thebasic peak contains an H chain C terminus with Lys at position 449 andan H chain C terminus with amidated Pro at position 447. The intensityof the basic peak was significantly reduced in the H chain C-terminalΔGK antibody, while no such significant reduction was observed in the Hchain C-terminal ΔK antibody. This suggests that the C-terminalheterogeneity of the H chain can be reduced only when the two C-terminalamino acids are deleted from the H chain.

The temperature of thermal denaturation of the H chain C-terminal ΔGKantibody was determined by DSC to assess the effect of the deletion ofthe two residues at the H chain C terminus on thermal stability. For theDSC measurement, the antibody was dialyzed against 20 mM acetic acidbuffer (pH 6.0) containing 150 mM NaCl to change the buffer. Afterthorough deaeration, the humanized PM-1 antibody and H chain C-terminalΔGK antibody solutions, and the reference solution (outer dialysate)were enclosed in calorimetric cells, and thoroughly thermallyequilibrated at 40° C. Then, the samples were scanned at from 40 to 100°C. with a rate of about 1K/min. The resulting denaturation peaks wereassigned (Rodolfo et al., Immunology Letters, 1999, p 47-52). The resultshowed that the C-terminal deletion had no effect on the thermaldenaturation temperature of CH3 domain.

Thus, the heterogeneity originated from the C-terminal amino acid can bereduced without affecting the thermal stability of antibody by deletingthe C-terminal lysine and glycine from the H chain constant region atthe nucleotide sequence level. Since all of the constant regions ofhuman antibodies IgG1, IgG2, and IgG4 contain Gly and Lys at positions446 and 447 in the EU numbering system in their C-terminal sequences,the method for reducing the C-terminal amino acid heterogeneitydiscovered in this example and others is also expected to be applicableto IgG2 and IgG4 constant regions.

Example 4 Construction of M14ΔGK with a Novel Optimized Constant RegionSequence

When an antibody pharmaceutical is aimed at neutralizing an antigen,effector functions such as ADCC of Fc domain are unnecessary andtherefore the binding to Fcγ receptor is unnecessary. The binding to Fcγreceptor is assumed to be unfavorable from the perspectives ofimmunogenicity and adverse effect (Non-patent Documents 5 and 6). Thehumanized anti-IL-6 receptor IgG1 antibody TOCILIZUMAB does not need tobind to Fcγ receptor, because it only needs to specifically bind to IL-6receptor and neutralize its biological activity in order to be used as atherapeutic agent for diseases associated with IL-6, such as rheumatoidarthritis.

Construction and Assessment of M14ΔGK, M11ΔGK, and M17ΔGK, FcγReceptor-nonbinding, Optimized Constant Regions

A possible method for impairing the Fcγ receptor binding is to convertthe IgG antibody from IgG1 isotype to IgG2 or IgG4 isotype (Ann.Hematol. 1998 June; 76(6):231-48). As a method for completelyeliminating the binding to Fcγ receptor, a method of introducing anartificial alteration into Fc domain has been reported. For example,since the effector functions of anti-CD3 antibody and anti-CD4 antibodycause adverse effects, amino acid mutations that are not present in thewild type sequence have been introduced into the Fcγ receptor-bindingregion of Fc domain (Non-patent Documents 3 and 7), and the resultingFcγ receptor-nonbinding anti-CD3 and anti-CD4 antibodies are currentlyunder clinical trials (Non-patent Documents 5 and 8). According toanother report (Patent Document 3), Fcγ receptor-nonbinding antibodiescan be prepared by converting the FcγR-binding domain of IgG1 (atpositions 233, 234, 235, 236, 327, 330, and 331 in the EU numberingsystem) into the sequence of IgG2 (at positions 233, 234, 235, and 236in the EU numbering system) or IgG4 (at positions 327, 330, and 331 inthe EU numbering system). However, if all of the above mutations areintroduced into IgG1, novel peptide sequences of nine amino acids, whichpotentially serve as non-natural T-cell epitope peptides, will begenerated, and this increases the immunogenicity risk. Theimmunogenicity risk should be minimized in developing antibodypharmaceuticals.

To overcome the above problem, alterations in the IgG2 constant regionwere considered. In the FcγR-binding domain of IgG2 constant region,residues at positions 327, 330, and 331 in the EU numbering system aredifferent from the nonbinding sequence of IgG4 while those at positions233, 234, 235, and 236 in the EU numbering system are amino acids ofnonbinding type. Thus, it is necessary to alter the amino acids atpositions 327, 330, and 331 in the EU numbering system to the sequenceof IgG4 (G2Δa described in Eur. J. Immunol. 1999 August; 29(8):2613-24).However, since the amino acid at position 339 in the EU numbering systemin IgG4 is alanine while the corresponding residue in IgG2 is threonine,a simple alteration of the amino acids at positions 327, 330, and 331 inthe EU numbering system to the sequence of IgG4 unfavorably generates anovel peptide sequence of 9 amino acids, potentially serving as anon-natural T-cell epitope peptide, and thus increases theimmunogenicity risk. Then, the present inventors found that thegeneration of novel peptide sequence could be prevented by introducingthe substitution of alanine for threonine at position 339 in the EUnumbering system in IgG2, in addition to the alteration described above.

In addition to the mutations described above, other mutations wereintroduced, and they were the substitution of valine for methionine atposition 397 in the EU numbering system in IgG2, which was discovered inExample 1 to improve the stability of IgG2 under acidic condition; andthe substitution of serine for cysteine at position 131 in the EUnumbering system, the substitution of lysine for arginine at position133 in the EU numbering system, and the substitution of serine forcysteine at position 219 in the EU numbering system, which werediscovered in Example 2 to improve the heterogeneity originated fromdisulfide bonds in the hinge region. Furthermore, since the mutations atpositions 131 and 133 generate a novel peptide sequence of 9 aminoacids, potentially serving as a non-natural T-cell epitope peptide, andthus generate the immunogenicity risk, the peptide sequence aroundpositions 131 to 139 was converted into a natural human sequence byintroducing the substitution of glycine for glutamic acid at position137 in the EU numbering system and the substitution of glycine forserine at position 138 in the EU numbering system. Furthermore, glycineand lysine at positions 446 and 447 in the EU numbering system weredeleted from the C terminus of H chain to reduce the C-terminalheterogeneity. The constant region sequence having all of the mutationsintroduced was named M14ΔGK (M14ΔGK, SEQ ID NO: 5). Although there is amutation of cysteine at position 219 to serine in M14ΔGK as a novel9-amino acid peptide sequence which potentially serves as a T-cellepitope peptide, the immunogenicity risk was considered very low sincethe amino acid property of serine is similar to that of cysteine. Theimmunogenicity prediction by TEPITOPE also suggested that there was nodifference in immunogenicity.

An expression vector for the antibody H chain sequence whose variableregion was WT and constant region was M14ΔGK (M14ΔGK, SEQ ID NO: 5;WT-M14ΔGK, SEQ ID NO: 19) was constructed by the method described inReference Example 1. An antibody having WT-M14ΔGK as H chain and WT as Lchain was expressed and purified by the method described in ReferenceExample 1.

Furthermore, in WT-M11ΔGK (M11ΔGK, SEQ ID NO: 8; WT-M11ΔGK, SEQ ID NO:21), mutations were introduced with the same method into the IgG4constant region at positions 233, 234, 235, and 236 in the EU numberingsystem (G44b described in Eur. J. Immunol. 1999 August; 29(8):2613-24;this alteration newly generates non-human sequence and thus increasesthe immunogenicity risk) to reduce the Fey receptor binding. In additionto the above alteration, to reduce the immunogenicity risk, mutationswere introduced at positions 131, 133, 137, 138, 214, 217, 219, 220,221, and 222 in the EU numbering system so that the pattern of disulfidebonding in the hinge region was the same as that of M14ΔGK; a mutationwas introduced at position 409 in the EU numbering system (Example 1) toimprove the stability under acidic condition; and the amino acids atpositions 446 and 447 in the EU numbering system were deleted (Example3) to reduce the C-terminal heterogeneity.

Furthermore, WT-M17ΔGK (M17ΔGK, SEQ ID NO: 10; WT-M17ΔGK, SEQ ID NO: 20)was constructed by introducing mutations into the IgG1 constant regionat positions 233, 234, 235, 236, 327, 330, 331, and 339 in the EUnumbering system (G1Δab described in Eur. J. Immunol. 1999 August;29(8):2613-24) to impair the Fcγ receptor binding and by deleting theamino acids at positions 446 and 447 in the EU numbering system toreduce the C-terminal heterogeneity (Example 3).

WT-M17ΔGK or WT-M11ΔGK was used as the H chain, and WT was used as the Lchain. These antibodies were expressed and purified by the methoddescribed in Example 1.

Assessment of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK for Fcγ ReceptorBinding

The FcγRI binding was assessed by the procedure described below. UsingBiacore T100, human-derived Fcγ receptor I (hereinafter FcγRI)immobilized onto a sensor chip was allowed to interact with IgG1, IgG2,IgG4, M11ΔGK, M14ΔGK, or M1ΔGK 7 as an analyte. The amounts of boundantibody were compared. The measurement was conducted using RecombinantHuman FcRIA/CD64 (R&D systems) as human-derived FcγRI, and IgG1, IgG2,IgG4, M11ΔGK, M14ΔGK, and M17ΔGK as samples. FcγRI was immobilized ontothe sensor chip CM5 (BIACORE) by the amine coupling method. The finalamount of immobilized hFcγRI was about 13000 RU. The running buffer usedwas HBS-EP+, and the flow rate was 20 μl/min. The sample concentrationwas adjusted to 100 μg/ml using HBS-EP+. The analysis included twosteps: two minutes of association phase where 10 μl of an antibodysolution was injected and the subsequent four minutes of dissociationphase where the injection was switched with HBS-EP+. After thedissociation phase, the sensor chip was regenerated by injecting 20 μlof 5 mM sodium hydroxide. The association, dissociation, andregeneration constitute one analysis cycle. Various antibody solutionswere injected to obtain sensorgrams. As analytes, IgG4, IgG2, IgG1, M11,M14, and M17 were injected in this order. This series of injection wasrepeated twice. The result of comparison of data on the determinedamounts of bound antibody is shown in FIG. 7. The comparison shows thatthe amount of bound antibody is reduced in the order of:IgG1>IgG4>>IgG2=M11ΔGK=M14ΔGK=M17ΔGK. Thus, it was revealed that theFcγRI binding of wild type IgG2, M11ΔGK, M14ΔGK, and M17ΔGK was weakerthan that of wild type IgG1 and IgG4.

The FcγRIIa binding was assessed by the procedure described below. UsingBiacore T100, human-derived Fcγ receptor IIa (hereinafter FcγRIIa)immobilized onto a sensor chip was allowed to interact with IgG1, IgG2,IgG4, M11ΔGK, M14ΔGK, or M17ΔGK as an analyte. The amounts of boundantibody were compared. The measurement was conducted using RecombinantHuman FcRIIA/CD32a (R&D systems) as human-derived FcγRIIa, and IgG1,IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK as samples. FcγRIIa wasimmobilized onto the sensor chip CM5 (BIACORE) by the amine couplingmethod. The final amount of immobilized FcγRIIa was about 3300 RU. Therunning buffer used was HBS-EP+, and the flow rate was 20 μl/min. Then,the running buffer was injected until the baseline was stabilized. Themeasurement was carried out after the baseline was stabilized. Theimmobilized FcγRIIa was allowed to interact with an antibody of each IgGisotype (IgG1, IgG2, or IgG4) or antibody introduced with mutations(M11ΔGK, M14ΔGK, or M17ΔGK) as an analyte. The amount of bound antibodywas observed. The running buffer used was HBS-EP+, and the flow rate was20 μl/min. The measurement temperature was 25° C. The concentration ofeach IgG or altered form thereof was adjusted to 100 μg/ml. 20 μl of ananalyte was injected and allowed to interact with the immobilizedFcγRIIa. After interaction, the analyte was dissociated from FcγRIIa andthe sensor chip was regenerated by injecting 200 μl of the runningbuffer. As analytes, IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK wereinjected in this order. This series of injection was repeated twice. Theresult of comparison of data on the amounts of bound antibody determinedis shown in FIG. 8. The comparison shows that the amount of boundantibody is reduced in the order of:IgG1>IgG2=IgG4>M11ΔGK=M14ΔGK=M17ΔGK. Thus, it was revealed that theFcγRIIa binding of M11ΔGK, M14ΔGK, and M17ΔGK was weaker than that ofwild type IgG1, IgG2, and IgG4.

The FcγRIIb binding was assessed by the procedure described below. UsingBiacore T100, human-derived Fcγ receptor IIb (hereinafter FcγRIIb)immobilized onto a sensor chip was allowed to interact with IgG1, IgG2,IgG4, M11ΔGK, M14ΔGK, or M17ΔGK as an analyte. The amounts of boundantibody were compared. The measurement was conducted using RecombinantHuman FcRIIB/C (R&D systems) as human-derived FcγRIIb, and IgG1, IgG2,IgG4, M11ΔGK, M14ΔGK, and M17ΔGK as samples. FcγRIIb was immobilizedonto the sensor chip CMS (BIACORE) by the amine coupling method. Thefinal amount of immobilized FcγRIIb was about 4300 RU. Then, the runningbuffer was injected until the baseline was stabilized. The measurementwas carried out after the baseline was stabilized. The immobilizedFcγRIIb was allowed to interact with an antibody of each IgG isotype(IgG1, IgG2, or IgG4) or antibody introduced with mutations (M11ΔGK,M14ΔGK, or M17ΔGK) as an analyte. The amount of bound antibody wasobserved. The running buffer used was HBS-EP+and the flow rate was 20μl/min. The measurement temperature was 25° C. The concentration of eachIgG or altered form thereof was adjusted to 200 μg/ml. 20 μl of ananalyte was injected and allowed to interact with the immobilizedFcγRIIb. After interaction, the analyte was dissociated from FcγRIIb andthe sensor chip was regenerated by injecting 200 μl of the runningbuffer. As analytes, IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK wereinjected in this order. This series of injection was repeated twice. Theresult of comparison of data on the amounts of bound antibody determinedis shown in FIG. 9. The comparison shows that the amount of boundantibody is reduced in the order of:IgG4>IgG1>IgG2>M11ΔGK=M14ΔGK=M17ΔGK. Thus, it was revealed that theFcγRIIb binding of M11ΔGK, M14ΔGK, and M17ΔGK was weaker than that ofwild type IgG1, IgG2, and IgG4.

The FcγRIIIa binding was assessed by the procedure described below.Using Biacore T100, human-derived Fcγ receptor IIIa (hereinafterFcγRIIIa) immobilized onto a sensor chip was allowed to interact withIgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, or M17ΔGK as an analyte. The amountsof bound antibody were compared. The measurement was conducted usinghFcγRIIIaV-His6 (recombinant hFcγRIIIaV-His6 prepared in the applicants'company) as human-derived FcγRIIIa, and IgG1, IgG2, IgG4, M11ΔGK,M14ΔGK, and M17ΔGK as samples. FcγRIIIa was immobilized onto the sensorchip CM5 (BIACORE) by the amine coupling method. The final amount ofimmobilized hFcγRIIIaV-His6 was about 8200 RU. The running buffer usedwas HBS-EP+, and the flow rate was 5 μl/min. The sample concentrationwas adjusted to 250 μg/ml using HBS-EP+. The analysis included twosteps: two minutes of association phase where 10 μl of an antibodysolution was injected and the subsequent four minutes of dissociationphase where the injection was switched with HBS-EP+. After thedissociation phase, the sensor chip was regenerated by injecting 20 μlof 5 mM hydrochloric acid. The association, dissociation, andregeneration constitute one analysis cycle. Various antibody solutionswere injected to obtain sensorgrams. As analytes, IgG4, IgG2, IgG1,M11ΔGK, M14ΔGK, and M17ΔGK were injected in this order. The result ofcomparison of data on the determined amounts of bound antibody is shownin FIG. 10. The comparison shows that the amount of bound antibody isreduced in the order of: IgG1>>IgG4>IgG2>M17ΔGK>M11ΔGK=M14ΔGK. Thus, itwas revealed that the FcγRIIIa binding of M11ΔGK, M14ΔGK, and M17ΔGK wasweaker than that of wild type IgG1, IgG2, and IgG4. Furthermore, theFcγRIIIa binding of M11ΔGK and M14ΔGK was found to be weaker than thatof M17ΔGK containing the mutation G1Δab reported in Eur. J. Immunol.1999 August; 29(8):2613-24.

The finding described above demonstrates that the Fcγ receptor bindingof WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK is markedly reduced as comparedto wild type IgG1. The immunogenicity risk due to Fcγ receptor-mediatedinternalization into APC and adverse effects caused by the effectorfunction such as ADCC can be avoided by using WT-M14ΔGK, WT-M17ΔGK, orWT-M11ΔGK as a constant region. Thus, WT-M14ΔGK, WT-M17ΔGK, andWT-M11ΔGK are useful as constant region sequence of antibodypharmaceuticals aimed at neutralizing antigens.

Assessment of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK for Stability at HighConcentrations

WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK were assessed for stability at highconcentrations. The purified antibodies of WT-IgG1, WT-M14ΔGK,WT-M17ΔGK, and WT-M11ΔGK were dialyzed against a solution of 20 mMhistidine chloride, 150 mM NaCl, pH 6.5 (EasySEP, TOMY), and thenconcentrated by ultrafilters. The antibodies were tested for stabilityat high concentrations. The conditions were as follows.

Antibodies: WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK

Buffer: 20 mM histidine chloride, 150 mM NaCl, pH 6.5

Concentration: 61 mg/ml

Storage temperature and time period: 40° C. for two weeks, 40° C. forone month, 40° C. for two months

Aggregation Assessment Method:

-   -   System: Waters Alliance    -   Column: G3000SWxl (TOSOH)    -   Mobile phase: 50 mM sodium phosphate, 300 mM KCl pH 7.0    -   Flow rate, wavelength: 0.5 ml/min, 220 nm    -   100 times diluted samples were analyzed

The contents of aggregate in the initial formulations (immediately afterpreparation) and formulations stored under various conditions wereestimated by gel filtration chromatography described above. Differences(amounts increased) in the content of aggregate relative to the initialformulations are shown in FIG. 11. The result showed that the amounts ofaggregate in WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK increased only slightlyas compared to WT-IgG1 and were about half of the content in WT.Furthermore, as shown in FIG. 12, the amount of increased Fab fragmentwas comparable between WT-IgG1 and WT-M17ΔGK, while the amountsincreased in WT-M14ΔGK and WT-M11ΔGK were about one quarter of theamount in WT. Degeneration pathways of IgG type antibody formulationsinclude formation of aggregate and generation of Fab degradate asdescribed in WO 2003/039485. Based on the two criteria, aggregation andFab fragment generation, WT-M14ΔGK and WT-M11ΔGK were demonstrated tohave a superior stability in formulations as compared to WT-IgG1. Thus,even for antibodies that have an IgG1 constant region with poorstability and could not be prepared as antibody pharmaceuticals inhigh-concentration liquid formulations, the use of WT-M14ΔGK, WT-M17ΔGK,or WT-M11ΔGK as a constant region was expected to allow production ofmore stable high-concentration liquid formulations.

In particular, M14ΔGK was expected to be very useful as a novel constantregion sequence that would (1) overcome the instability of the originalIgG2 molecule under acidic condition; (2) improve the heterogeneityoriginated from disulfide bonds in the hinge region; (3) not bind to Fcγreceptor; (4) have a minimized number of novel peptide sequences of 9amino acids which potentially serve as T-cell epitope peptides; and (5)have a better stability than IgG1 in high-concentration formulations.

Example 5 Preparation and Assessment of M31ΔGK

M14ΔGK prepared in Example 4 was altered by substituting the IgG2sequence for the amino acids at positions 330, 331, and 339 in the EUnumbering system to construct M31ΔGK (M31ΔGK, SEQ ID NO: 7). Anexpression vector for a sequence of antibody H chain whose variableregion is WT and constant region sequence is M31ΔGK (WT-M31ΔGK, SEQ IDNO: 22) was constructed by the method described in Reference Example 1.Using WT-M31ΔGK H chain and WT L chain, WT-M31ΔGK was expressed andpurified by the method described in Reference Example 1.

In addition to WT-M31ΔGK, WT-IgG2 and WT-M14ΔGK were expressed andpurified at the same time, and analyzed by cation exchangechromatography by the procedure described below. The conditions used inthe cation exchange chromatography analysis were as follows.Chromatograms for WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK were compared.

-   -   Column: ProPac WCX-10 (Dionex)    -   Mobile phase A: 25 mmol/l MES/NaOH, pH 6.1        -   B: 25 mmol/l MES/NaOH, 250 mmol/l NaCl, pH 6.1    -   Flow rate: 0.5 ml/min    -   Gradient: 0% B (5 min)->(65 min)->100% B->(1 min)    -   Detection: 280 nm

The analysis result for WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK is shown inFIG. 13. Like WT-M14ΔGK, WT-M31ΔGK was demonstrated to be eluted as asingle peak, while WT-IgG2 gave multiple peaks. This indicates that theheterogeneity derived from disulfide bonds in the hinge region of IgG2can also be avoided in WT-M31ΔGK.

Example 6 Assessment of the Plasma Retention of WT-M14

Method for Estimating the Retention in Human Plasma

The prolonged retention (slow elimination) of IgG molecule in plasma isknown to be due to the function of FcRn which is known as a salvagereceptor of IgG molecule (Nat. Rev. Immunol. 2007 September;7(9):715-25). When taken up into endosomes via pinocytosis, under theacidic conditions within endosome (approx. pH 6.0), IgG molecules bindto FcRn expressed in endosomes. While IgG molecules that do not bind toFcRn are transferred and degraded in lysosomes, those bound to FcRn aretranslocated to the cell surface and then released from FcRn back intoplasma again under the neutral conditions in plasma (approx. pH 7.4).

Known IgG-type antibodies include the IgG1, IgG2, IgG3, and IgG4isotypes. The plasma half-lives of these isotypes in human are reportedto be about 36 days for IgG1 and IgG2; about 29 days for IgG3; and 16days for IgG4 (Nat. Biotechnol. 2007 December; 25(12):1369-72). Thus,the retention of IgG1 and IgG2 in plasma is believed to be the longest.In general, the isotypes of antibodies used as pharmaceutical agents areIgG1, IgG2, and IgG4. Methods reported for further improving thepharmacokinetics of these IgG antibodies include methods for improvingthe above-described binding to human FcRn, and this is achieved byaltering the sequence of IgG constant region (J. Biol. Chem. 2007 Jan.19; 282(3):1709-17; J. Immunol. 2006 Jan. 1; 176(1):346-56).

There are species-specific differences between mouse FcRn and human FcRn(Proc. Natl. Acad. Sci. USA. 2006 Dec. 5; 103(49):18709-14). Therefore,to predict the plasma retention of IgG antibodies that have an alteredconstant region sequence in human, it is desirable to assess the bindingto human FcRn and retention in plasma in human FcRn transgenic mice(Int. Immunol. 2006 December; 18(12):1759-69).

Assessment of the Binding to Human FcRn

FcRn is a complex of FcRn and β2-microglobulin. Oligo-DNA primers wereprepared based on the human FcRn gene sequence disclosed (J. Exp. Med.(1994) 180 (6), 2377-2381). A DNA fragment encoding the whole gene wasprepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA,Clontech) as a template and the prepared primers. Using the obtained DNAfragment as a template, a DNA fragment encoding the extracellular domaincontaining the signal region (Metl-Leu290) was amplified by PCR, andinserted into an animal cell expression vector (the amino acid sequenceof human FcRn as set forth in SEQ ID NO: 24). Likewise, oligo-DNAprimers were prepared based on the human β2-microglobulin gene sequencedisclosed (Proc. Natl. Acad. Sci. USA. (2002) 99 (26), 16899-16903). ADNA fragment encoding the whole gene was prepared by PCR using humancDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) as a template and theprepared primers. Using the obtained DNA fragment as a template, a DNAfragment encoding the whole β2-microglobulin containing the signalregion (Met1-Met119) was amplified by PCR and inserted into an animalcell expression vector (the amino acid sequence of humanβ2-microglobulin as set forth in SEQ ID NO: 25).

Soluble human FcRn was expressed by the following procedure. Theplasmids constructed for human FcRn and β2-microglobulin were introducedinto cells of the human embryonic kidney cancer-derived cell lineHEK293H (Invitrogen) using 10% Fetal Bovine Serum (Invitrogen) bylipofection. The resulting culture supernatant was collected, and FcRnwas purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences) bythe method described in J. Immunol. 2002 Nov. 1; 169(9):5171-80,followed by further purification using HiTrap Q HP (GE Healthcare).

The binding to human FcRn was assessed using Biacore 3000. An antibodywas bound to Protein L or rabbit anti-human IgG Kappa chain antibodyimmobilized onto a sensor chip, human FcRn was added as an analyte forinteraction with the antibody, and the affinity (KD) was calculated fromthe amount of bound human FcRn. Specifically, Protein L or rabbitanti-human IgG Kappa chain antibody was immobilized onto sensor chip CMS(BIACORE) by the amine coupling method using 50 mM Na-phosphate buffer(pH 6.0) containing 150 mM NaCl as the running buffer. Then, an antibodywas diluted with a running buffer containing 0.02% Tween20, and injectedto be bound to the chip. Human FcRn was then injected and the binding ofthe human FcRn to antibody was assessed.

The affinity was computed using BIAevaluation Software. The obtainedsensorgram was used to calculate the amount of hFcRn bound to theantibody immediately before the end of human FcRn injection. Theaffinity of the antibody for human FcRn was calculated by fitting withthe steady state affinity method.

Assessment for the Plasma Retention in Human FcRn Transgenic Mice

The pharmacokinetics in human FcRn transgenic mice (B6.mFcRn−/−.hFcRn Tgline 276 +/+ mice; Jackson Laboratories) was assessed by the followingprocedure. An antibody was intravenously administered once at a dose of1 mg/kg to mice, and blood was collected at appropriate time points. Thecollected blood was immediately centrifuged at 15,000 rpm and 4° C. for15 minutes to obtain blood plasma. The separated plasma was stored in afreezer at −20° C. or below until use. The plasma concentration wasdetermined by ELISA.

Predictive Assessment of the Plasma Retention of WT-M14 in Human

The bindings of WT-IgG1 and WT-M14 to human FcRn were assessed byBIAcore. As shown in Table 1, the result indicated that the binding ofWT-M14 was slightly greater than that of WT-IgG1.

TABLE 1 KD (μM) WT-IgG1 2.07 WT-M14 1.85

As shown in FIG. 14, however, the retention in plasma was comparablebetween WT-IgG1 and WT-M14 when assessed using human FcRn transgenicmice. This finding suggests that the plasma retention of the M14constant region in human is comparable to that of the IgG1 constantregion.

Example 7 Preparation of WT-M44, WT-M58, and WT-M73 which have ImprovedPharmacokinetics

Preparation of the WT-M58 Molecule

As described in Example 6, the plasma retention of WT-M14 in human FcRntransgenic mice was comparable to that of WT-IgG1. Known methods toimprove pharmacokinetics include those to lower the isoelectric point ofan antibody and those to enhance the binding to FcRn. Here, themodifications described below were introduced to improve thepharmacokinetics of WT-M14. Specifically, the following substitutionswere introduced into WT-M31ΔGK, which was prepared from WT-M14 asdescribed in Example 4: substitution of methionine for valine atposition 397; substitution of glutamine for histidine at position 268;substitution of glutamine for arginine at position 355; and substitutionof glutamic acid for glutamine at position 419 in the EU numberingsystem. These four substitutions were introduced into WT-M31ΔGK togenerate WT-M58 (amino acid sequence of SEQ ID NO: 26). Expressionvectors were prepared by the same method described in Example 1. WT-M58and L(WT) were used as H chain and L chain, respectively. WT-M58 wasexpressed and purified by the method described in Example 1.

Construction of the WT-M73 Molecule

On the other hand, WT-M44 (amino acid sequence of SEQ ID NO: 27) wasgenerated by introducing into IgG1 a substitution of alanine for theamino acid at position 434, EU numbering. WT-M83 (amino acid sequence ofSEQ ID NO: 58) was also generated by deletions of glycine at position446, EU numbering and lysine at position 447, EU numbering to reduce Hchain C-terminal heterogeneity. Furthermore, WT-M73 (amino acid sequenceof SEQ ID NO: 28) was generated by introducing into WT-M58 asubstitution of alanine at position 434, EU numbering.

Expression vectors for the above antibodies were constructed by themethod described in Example 1. WT-M44, WT-M58, or WT-M73 was used as Hchain, while L (WT) was used as L chain. WT-M44, WT-M58, and WT-M73 wereexpressed and purified by the method described in Example 1.

Predictive Assessment of the Plasma Retention of WT-M44, WT-M58, andWT-M73 in Human

The bindings of WT-IgG1, WT-M44, WT-M58, and WT-M73 to human FcRn wereassessed by BIAcore. As shown in Table 2, the result indicates that thebindings of WT-M44, WT-M58, and WT-M73 are greater than WT-IgG1, andabout 2.7, 1.4, and 3.8 times of that of WT-IgG1, respectively.

TABLE 2 KD (μM) WT-IgG1 1.62 WT-M44 0.59 WT-M58 1.17 WT-M73 0.42

As a result of assessing WT-IgG1, WT-M14, and WT-M58 for their plasmaretention in human FcRn transgenic mice, as shown in FIG. 24, WT-M58 wasconfirmed to have increased retention in plasma relative to WT-IgG1 andWT-M14. Furthermore, WT-IgG1, WT-M44, WT-M58, and WT-M73 were assessedfor their plasma retention in human FcRn transgenic mice. As shown inFIG. 15, all of WT-M44, WT-M58, and WT-M73 were confirmed to haveimproved pharmacokinetics relative to WT-IgG1. Thepharmacokinetics-improving effect correlated with the binding activityto human FcRn. In particular, the plasma level of WT-M73 at Day 28 wasimproved to about 16 times of that of WT-IgG1. This finding suggeststhat the pharmacokinetics of antibodies with the M73 constant region inhuman is also significantly enhanced when compared to antibodies withthe IgG1 constant region.

Example 8 Effect of the Novel Constant Regions M14 and M58 in ReducingHeterogeneity in Various Antibodies

As described in Example 4, it was demonstrated that the heterogeneityoriginated from the hinge region of IgG2 could be reduced by convertingthe IgG2 constant region to M14 in the humanized anti-IL-6 receptor PM1antibody (WT). IgG2 type antibodies other than the humanized PM1antibody were also tested to assess whether the heterogeneity can bereduced by converting their constant regions into M14 or M58.

Antibodies other than the humanized PM1 antibody were: the anti IL-6receptor antibody F2H/L39 (the amino acid sequences of F2H/L39_VH andF2H/L39 VL as set forth in SEQ ID NOs: 29 and 30, respectively);anti-IL-31 receptor antibody H0L0 (the amino acid sequences of H0L0_VHand H0L0_VL as set forth in SEQ ID NOs: 31 and 32, respectively); andanti-RANKL antibody DNS (the amino acid sequences of DNS_VH and DNS_VLas set forth in SEQ ID NOs: 33 and 34, respectively). For each of theseantibodies, antibodies with IgG1 constant region (SEQ ID NO: 1), IgG2constant region (SEQ ID NO: 2), or M14 (SEQ ID NO: 5) or M58 (SEQ ID NO:35) were generated.

The generated antibodies were assessed for heterogeneity by cationexchange chromatography using an adequate gradient and an appropriateflow rate on a ProPac WCX-10 (Dionex) column (mobile phase A: 20 mMsodium acetate (pH 5.0), mobile phase B: 20 mM sodium acetate/1M NaCl(pH 5.0)). The assessment result obtained by cation exchangechromatography (IEC) is shown in FIG. 16.

As shown in FIG. 16, conversion of the constant region from an IgG1 typeinto an IgG2 type was demonstrated to increase heterogeneity not only inthe humanized anti-IL-6 receptor PM1 antibody (WT), but also in theanti-IL-6 receptor antibody F2H/L39, anti-IL-31 receptor antibody H0L0,and anti-RANKL antibody DNS. In contrast, heterogeneity could bedecreased in all of these antibodies by converting their constant regioninto M14 or M58. Thus, it was demonstrated that, regardless of the typeof antigen or antibody variable region sequence, the heterogeneityoriginated from natural IgG2 could be reduced by substituting serinesfor cysteines at position 131, EU numbering, in the H-chain CH1 domainand at position 219, EU numbering, in the upper hinge of H chain.

Example 9 Effect of the Novel Constant Region M58 to Improve thePharmacokinetics in Various Antibodies

As described in Example 7, it was demonstrated that conversion of theconstant region from IgG1 into M58 in the humanized anti-IL-6 receptorPM1 antibody (WT) improved the binding to human FcRn andpharmacokinetics in human FcRn transgenic mice. So, IgG1 type antibodiesother than the humanized PM1 antibody were also tested to assess whethertheir pharmacokinetics can be improved by converting their constantregion into M58.

Antibodies other than the humanized PM1 antibody (WT) were theanti-IL-31 receptor antibody H0L0 (the amino acid sequences of H0L0_VHand H0L0_VL as set forth in SEQ ID NOs: 31 and 32, respectively) andanti-RANKL antibody DNS (the amino acid sequences of DNS_VH and DNS_VLas set forth in SEQ ID NOs: 33 and 34, respectively). For each of theseantibodies, antibodies with IgG1 constant region (SEQ ID NO: 1) or M58(SEQ ID NO: 35) were generated and assessed for their binding to humanFcRn by the method described in Example 6. The result is shown in Table3.

TABLE 3 KD (μM) WT H0L0 DNS IgG1 1.42 1.07 1.36 M58 1.03 0.91 1.03

As shown in Table 3, it was demonstrated that as a result of conversionof the constant region from the IgG1 type to M58, as with anti-IL-6receptor antibody WT, the bindings of both the anti-IL-31 receptorantibody H0L0 and anti-RANKL antibody DNS to human FcRn were improved.This suggests the possibility that regardless of the type of antigen orsequence of antibody variable region, the pharmacokinetics in human isimproved by converting the constant region from the IgG1 type to M58.

Example 10 Effect of Cysteine in the CH1 Domain on Heterogeneity andStability

As described in Example 2, cysteines in the hinge region and CH1 domainof IgG2 were substituted to decrease the heterogeneity of natural IgG2.Assessment of various altered antibodies revealed that heterogeneitycould be reduced without decreasing stability by using SKSC (SEQ ID NO:38). SKSC (SEQ ID NO: 38) is an altered constant region obtained bysubstituting serine for cysteine at position 131 and lysine for arginineat position 133, EU numbering, in the H-chain CH1 domain, and serine forcysteine at position 219, EU numbering, in the H-chain upper hinge ofthe wild type IgG2 constant region sequence.

Meanwhile, another possible method for decreasing heterogeneity is asingle substitution of serine for cysteine at position 219, or serinefor cysteine at position 220, EU numbering, in the H-chain upper hinge.The altered IgG2 constant region SC (SEQ ID NO: 39) was prepared bysubstituting serine for cysteine at position 219 and CS (SEQ ID NO: 40)was prepared by substituting serine for cysteine at position 220, EUnumbering, in IgG2. WT-SC (SEQ ID NO: 41) and WT-CS (SEQ ID NO: 42) wereprepared to have SC and CS, respectively, and compared with WT-IgG1,WT-IgG2, WT-SKSC, and WT-M58 in terms of heterogeneity and thermalstability. Furthermore, F2H/L39-IgG1, F2H/L39-IgG2, F2H/L39-SC,F2H/L39-CS, F2H/L39-SKSC, and F2H/L39-M14, which have the constantregion of IgG1 (SEQ ID NO: 1), IgG2 (SEQ ID NO: 2), SC (SEQ ID NO: 39),CS (SEQ ID NO: 40), SKSC (SEQ ID NO: 38), or M14 (SEQ ID NO: 5),respectively, were prepared from F2H/L39 (the amino acid sequences ofF2H/L39_VH and F2H/L39_VL as set forth in SEQ ID NOs: 29 and 30,respectively), which is an anti IL-6 receptor antibody different fromWT. The antibodies were compared with regard to heterogeneity.

WT-IgG1, WT-IgG2, WT-SC, WT-CS, WT-SKSC, WT-M58, F2H/L39-IgG1,F2H/L39-IgG2, F2H/L39-SC, F2H/L39-CS, F2H/L39-SKSC, and F2H/L39-M14 wereassessed for heterogeneity by cation exchange chromatography using anadequate gradient and an appropriate flow rate on a ProPac WCX-10(Dionex) column (mobile phase A: 20 mM sodium acetate (pH 5.0), mobilephase B: 20 mM sodium acetate/1M NaCl (pH 5.0)). The assessment resultobtained by cation exchange chromatography is shown in FIG. 17.

As shown in FIG. 17, conversion of the constant region from an IgG1 typeto an IgG2 type was demonstrated to increase heterogeneity in both WTand F2H/L39. In contrast, heterogeneity was significantly decreased byconverting the constant region into SKSC and M14 or M58. Meanwhile,conversion of the constant region into SC significantly decreasedheterogeneity, as in the case of SKSC. However, conversion into CS didnot sufficiently improve heterogeneity.

In addition to low heterogeneity, high stability is generally desiredwhen preparing stable formulations in development of antibodypharmaceuticals. Thus, to assess stability, the midpoint temperature ofthermal denaturation (Tm value) was determined by differential scanningcalorimetry (DSC) (VP-DSC; Microcal). The midpoint temperature ofthermal denaturation (Tm value) serves as an indicator of stability. Inorder to prepare stable formulations as pharmaceutical agents, a highermidpoint temperature of thermal denaturation (Tm value) is preferred (J.Pharm. Sci. 2008 April; 97(4):1414-26). WT-IgG1, WT-IgG2, WT-SC, WT-CS,WT-SKSC, and WT-M58 were dialyzed (EasySEP; TOMY) against a solution of20 mM sodium acetate, 150 mM NaCl, pH 6.0. DSC measurement was carriedout at a heating rate of 1° C./min in a range of 40 to 100° C., and at aprotein concentration of about 0.1 mg/ml. The denaturation curvesobtained by DSC are shown in FIG. 18. The Tm values of the Fab domainsare listed in Table 4 below.

TABLE 4 Tm/° C. WT-IgG1 94.8 WT-IgG2 93.9 WT-SC 86.7 WT-CS 86.4 WT-SKSC93.7 WT-M58 93.7

The Tm values of WT-IgG1 and WT-IgG2 were almost the same (about 94° C.;Tm of IgG2 was about 1° C. lower). Meawhile, the Tm values of WT-SC andWT-CS were about 86° C., and thus significantly lower than those ofWT-IgG1 and WT-IgG2. On the other hand, the Tm values of WT-M58 andWT-SKSC were about 94° C., and comparable to those of WT-IgG1 andWT-IgG2. This suggests that WT-SC and WT-CS are markedly unstable ascompared to IgG2, and thus, WT-SKSC and WT-M58, both of which alsocomprise substituion of serine for cysteine in the CH1 domain, arepreferred in the development of antibody pharmaceuticals. The reason forthe significant decrease of Tm in WT-SC and WT-CS relative to IgG2 isthought to be differences in the disulfide-bonding pattern between WT-SCor WT-CS and IgG2.

Furthermore, comparison of DSC denaturation curves showed that WT-IgG1,WT-SKSC, and WT-M58 each gave a sharp and single denaturation peak forthe Fab domain. In contrast, WT-SC and WT-CS each gave a broaderdenaturation peak for the Fab domain. WT-IgG2 also gave a shoulder peakon the lower temperature side of the Fab domain denaturation peak. Ingeneral, it is considered that a single component gives a sharp DSCdenaturation peak, and when two or more components with different Tmvalues (namely, heterogeneity) are present, the denaturation peakbecomes broader. Specifically, the above-described result suggests thepossibility that each of WT-IgG2, WT-SC, and WT-CS contains two or morecomponents, and thus the natural-IgG2 heterogeneity has not beensufficiently reduced in WT-SC and WT-CS. This finding suggests that notonly cysteines in the hinge region but also those in the CH1 domain areinvolved in the wild type-IgG2 heterogeneity, and it is necessary toalter not only cysteines in the hinge region but also those in the CH1domain to decrease the DSC heterogeneity. Furthermore, as describedabove, stability comparable to that of wild type IgG2 can be acheivedonly when cysteines in both the hinge region and CH1 domain aresubstituted.

The above finding suggests that from the perspective of heterogeneityand stability, SC and CS, which are constant regions introduced withserine substitution for only the hinge region cysteine, are insufficientas constant regions to decrease heterogeneity originated from the hingeregion of IgG2. It was thus discovered that the heterogeneity could besignificantly decreased while maintaining an IgG2-equivalent stability,only when the cysteine at position 131, EU numbering, in the CH1 domainwas substituted with serine in addition to cysteine at hinge region.Such constant regions include M14, M31, M58, and M73 described above. Inparticular, M58 and M73 are stable and less heterogeneous, and exhibitimproved pharmacokinetics, and therefore are expected to be very usefulas constant regions for antibody pharmaceuticals.

Example 11 Generation of Fully Humanized anti-IL-6 Receptor Antibodieswith Improved PK/PD

To generate a fully humanized anti-IL-6 receptor antibody with improvedPK/PD, the molecules described below were created by alteringTOCILIZUMAB (H chain, WT-IgG1 (SEQ ID NO: 12); L chain, WT (SEQ ID NO:15). The following fully humanized IL-6 receptor antibodies wereprepared which use as constant region M73 or M83 prepared in Example 7:Fv3-M73 (H chain, VH4-M73, SEQ ID NO: 48; L chain, VL1-kappa, SEQ ID NO:49), Fv4-M73 (H chain, VH3-M73, SEQ ID NO: 46; L chain, VL3-kappa, SEQID NO: 47), and Fv5-M83 (H chain, VH5-M83, SEQ ID NO: 44; L chain,VL5-kappa, SEQ ID NO: 45).

The affinities of prepared Fv3-M73, Fv4-M73, and Fv5-M83 against IL-6receptor were compared to that of TOCILIZUMAB. The affinities of theseanti-IL-6 receptor antibodies determined are shown in Table 5 (seeReference Example for method). Furthermore, their BaF/gp130-neutralizingactivities were compared to those of TOCILIZUMAB and the control (theknown high affinity anti-IL-6 receptor antibody described in ReferenceExample, and VQ8F11-21 hIgG1 described in US 2007/0280945) (seeReference Example for method). The results obtained by determining thebiological activities of these antibodies using BaF/gp130 are shown inFIG. 19 (TOCILIZUMAB, the control, and Fv5-M83 with a final IL-6concentration of 300 ng/ml) and FIG. 20 (TOCILIZUMAB, Fv3-M73, andFv4-M73 with a final IL-6 concentration of 30 ng/ml). As shown in Table5, Fv3-M73 and Fv4-M73 have about two to three times higher affinitythan TOCILIZUMAB, while Fv5-M83 exhibits about 100 times higher affinitythan TOCILIZUMAB (since it was difficult to measure the affinity ofFv5-M83, instead the affinity was determined using Fv5-IgG1, which hasan IgG1-type constant region; the constant region is generally thoughtto have no effect on affinity). As shown in FIG. 20, Fv3-M73 and Fv4-M73exhibit slightly stronger activities than TOCILIZUMAB. As shown in FIG.19, Fv5-M83 has a very strong activity, which is more than 100 timesgreater than that of TOCILIZUMAB in terms of 50% inhibitoryconcentration. Fv5-M83 also exhibits about 10 times higher neutralizingactivity in terms of 50% inhibitory concentration than the control (theknown high-affinity anti-IL-6 receptor antibody).

TABLE 5 k_(a) (1/Ms) k_(d) (1/s) KD (M) TOCILIZUMAB 4.0E+05 1.1E−032.7E−09 Fv3-M73 8.5E+05 8.7E−04 1.0E−09 Fv4-M73 7.5E+05 1.0E−03 1.4E−09Fv5-M83 1.1E+06 2.8E−05 2.5E−11

The isoelectric points of TOCILIZUMAB, the control, Fv3-M73, Fv4-M73,and Fv5-M83 were determined by isoelectric focusing using a method knownto those skilled in the art. The result showed that the isoelectricpoint was about 9.3 for TOCILIZUMAB; about 8.4 to 8.5 for the control;about 5.7 to 5.8 for Fv3-M73; about 5.6 to 5.7 for Fv4-M73; and 5.4 to5.5 for Fv5-M83. Thus, each antibody had a significantly loweredisoelectric point when compared to TOCILIZUMAB and the control.Furthermore, the theoretical isoelectric point of the variable regionsVH/VL was calculated by GENETYX (GENETYX CORPORATION). The result showedthat the theoretical isoelectric point was 9.20 for TOCILIZUMAB; 7.79for the control; 5.49 for Fv3-M73; 5.01 for Fv4-M73; and 4.27 forFv5-M83. Thus, each antibody had a significantly lowered isoelectricpoint when compared to TOCILIZUMAB and the control. Accordingly, thepharmacokinetics of Fv3-M73, Fv4-M73, and Fv5-M83 was thought to beimproved when compared to TOCILIZUMAB and the control.

T-cell epitopes in the variable region sequence of TOCILIZUMAB, Fv3-M73,Fv4-M73, or Fv5-M83 were analyzed using TEPITOPE (Methods. 2004December; 34(4):468-75). As a result, TOCILIZUMAB was predicted to haveT-cell epitopes, of which many could bind to HLA. In contrast, thenumber of sequences that were predicted to bind to T-cell epitopes wassignificantly reduced in Fv3-M73, Fv4-M73, and Fv5-M83. In addition, theframework of Fv3-M73, Fv4-M73, or Fv5-M83 has no mouse sequence and isthus fully humanized. These suggest the possibility that immunogenicityrisk is significantly reduced in Fv3-M73, Fv4-M73, and Fv5-M83 whencompared to TOCILIZUMAB.

Example 12 PK/PD Test of Fully Humanized Anti-IL-6 Receptor Antibodiesin Monkeys

Each of TOCILIZUMAB, the control, Fv3-M73, Fv4-M73, and Fv5-M83 wasintravenously administered once at a dose of 1 mg/kg to cynomolgusmonkeys to assess the time courses of their plasma concentrations (seeReference Example for method). The plasma concentration time courses ofTOCILIZUMAB, Fv3-M73, Fv4-M73, and Fv5-M83 after intravenousadministration are shown in FIG. 21. The result showed that each ofFv3-M73, Fv4-M73, and Fv5-M83 exhibited significantly improvedpharmacokinetics in cynomolgus monkeys when compared to TOCILIZUMAB andthe control. Of them, Fv3-M73 and Fv4-M73 exhibited substantiallyimproved pharmacokinetics when compared to TOCILIZUMAB.

The efficacy of each antibody to neutralize membrane-bound cynomolgusmonkey IL-6 receptor was assessed. Cynomolgus monkey IL-6 wasadministered subcutaneously in the lower back at 5 μg/kg every day fromDay 6 to Day 18 after antibody administration (Day 3 to Day 10 forTOCILIZUMAB), and the CRP concentration in each animal was determined 24hours later (see Reference Example for method). The time course of CRPconcentration after administration of each antibody is shown in FIG. 22.To assess the efficacy of each antibody to neutralize soluble cynomolgusmonkey IL-6 receptor, the plasma concentration of free solublecynomolgus monkey IL-6 receptor in the cynomolgus monkeys was determinedand percentage of soluble IL-6 receptor neutralization were calculated(see Reference Example for method). The time course of percentage ofsoluble IL-6 receptor neutralization after administration of eachantibody is shown in FIG. 23.

Each of Fv3-M73, Fv4-M73, and Fv5-M83 neutralized membrane-boundcynomolgus monkey IL-6 receptor in a more sustainable way, andsuppressed the increase of CRP over a longer period when compared toTOCILIZUMAB and the control (the known high-affinity anti-IL-6 receptorantibody). Furthermore, each of Fv3-M73, Fv4-M73, and Fv5-M83neutralized soluble cynomolgus monkey IL-6 receptor in a moresustainable way, and suppressed the increase of free soluble cynomolgusmonkey IL-6 receptor over a longer period when compared to TOCILIZUMABand the control. These findings demonstrate that all of Fv3-M73,Fv4-M73, and Fv5-M83 are superior in sustaining the neutralization ofmembrane-bound and soluble IL-6 receptors than TOCILIZUMAB and thecontrol. Of them, Fv3-M73 and Fv4-M73 are remarkably superior insustaining the neutralization. Meanwhile, Fv5-M83 suppressed CRP andfree soluble cynomolgus monkey IL-6 receptor more strongly than Fv3-M73and Fv4-M73. Thus, Fv5-M83 is considered to be stronger than Fv3-M73,Fv4-M73, and the control (the known high-affinity anti-IL-6 receptorantibody) in neutralizing membrane-bound and soluble IL-6 receptors. Itwas considered that results in in vivo of cynomolgus monkeys reflect thestronger affinity of Fv5-M83 for IL-6 receptor and stronger biologicalactivity of Fv5-M83 in the BaF/gp130 assay system relative to thecontrol.

These findings suggest that Fv3-M73 and Fv4-M73 are highly superior insustaining their activities as an anti-IL-6 receptor-neutralizingantibody when compared to TOCILIZUMAB and the control, and thus enableto significantly reduce the dosage and frequency of administration.Furthermore, Fv5-M83 was demonstrated to be remarkably superior in termsof the strength of activity as an anti-IL-6 receptor-neutralizingantibody as well as sustaining their activity. Thus, Fv3-M73, Fv4-M73,and Fv5-M83 are expected to be useful as pharmaceutical IL-6antagonists.

Reference Example

Preparation of Soluble Recombinant Cynomolgus Monkey IL-6 Receptor(cIL-6R)

Oligo-DNA primers were prepared based on the disclosed gene sequence forRhesus monkey IL-6 receptor (Birney et al., Ensembl 2006, Nucleic AcidsRes. 2006 Jan. 1; 34 (Database issue):D556-61). A DNA fragment encodingthe whole cynomolgus monkey IL-6 receptor gene was prepared by PCR usingthe primers, and as a template, cDNA prepared from the pancreas ofcynomolgus monkey. The resulting DNA fragment was inserted into ananimal cell expression vector, and a stable expression CHO line(cyno.sIL-6R-producing CHO cell line) was prepared using the vector. Theculture medium of cyno.sIL-6R-producing CHO cells was purified using aHisTrap column (GE Healthcare Bioscience) and then concentrated withAmicon Ultra-15 Ultracel-10k (Millipore). A final purified sample ofsoluble cynomolgus monkey IL-6 receptor (hereinafter cIL-6R) wasobtained through further purification on a Superdex 200 pg 16/60 gelfiltration column (GE Healthcare Bioscience).

Preparation of Recombinant Cynomolgus Monkey IL-6 (cIL-6)

Cynomolgus monkey IL-6 was prepared by the procedure described below.The nucleotide sequence encoding 212 amino acids deposited underSWISSPROT Accession No. P79341 was prepared and cloned into an animalcell expression vector. The resulting vector was introduced into CHOcells to prepare a stable expression cell line (cyno.IL-6-producing CHOcell line). The culture medium of cyno.IL-6-producing CHO cells waspurified using a SP-Sepharose/FF column (GE Healthcare Bioscience) andthen concentrated with Amicon Ultra-15 Ultracel-5k (Millipore). A finalpurified sample of cynomolgus monkey IL-6 (hereinafter cIL-6) wasobtained through further purification on a Superdex 75 pg 26/60 gelfiltration column (GE Healthcare Bioscience), followed by concentrationwith Amicon Ultra-15 Ultracel-5k (Millipore).

Preparation of a known High-affinity Anti-IL-6 Receptor Antibody

An animal cell expression vector was constructed to express VQ8F11-21hIgG1, a known high-affinity anti-IL-6 receptor antibody. VQ8F11-21hIgG1 is described in US 2007/0280945 A1 (US 2007/0280945 A1; the aminoacid sequences of H chain and L chain as set forth in SEQ ID NOs: 19 and27, respectively). The antibody variable region was constructed by PCRusing a combination of synthetic oligo DNAs (assembly PCR). IgG1 wasused as the constant region. The antibody variable and constant regionswere combined together by assembly PCR, and then inserted into an animalcell expression vector to construct expression vectors for the H chainand L chain of interest. The nucleotide sequences of the resultingexpression vectors were determined by a method known to those skilled inthe art. The high-affinity anti-IL-6 receptor antibody (hereinafterabbreviated as “control”) was expressed and purified using theconstructed expression vectors by the method described in Example 1.

Assessment for the Biological Activity by Human gp130-expressing BaF3Cells (BaF/gp130)

The IL-6 receptor neutralizing activity was assessed using BaF3/gp130which proliferates in an IL-6/IL-6 receptor-dependent manner. Afterthree washes with RPMI1640 supplemented with 10% FBS, BaF3/gp130 cellswere suspended at 5×10⁴ cells/ml in RPMI1640 supplemented with 600 ng/mlor 60 ng/ml human interleukin-6 (TORAY) (final concentration of 300ng/ml or 30 ng/ml, respectively), appropriate amount of recombinantsoluble human IL-6 receptor (SR344), and 10% FBS. The cell suspensionswere dispensed (50 μl/well) into 96-well plates (CORNING). Then, thepurified antibodies were diluted with RPMI1640 containing 10% FBS, andadded to each well (50 μl/well). The cells were cultured at 37° C. under5% CO₂ for three days. WST-8 Reagent (Cell Counting Kit-8; DojindoLaboratories) was diluted two-fold with PBS. Immediately after 20 μl ofthe reagent was added to each well, the absorbance at 450 nm (referencewavelength: 620 nm) was measured using SUNRISE CLASSIC (TECAN). Afterculturing for two hours, the absorbance at 450 nm (reference wavelength:620 nm) was measured again. The IL-6 receptor neutralizing activity wasassessed using the change of absorbance during two hours as anindicator.

Biacore-based Analysis of Binding to IL-6 Receptor

Antigen-antibody reaction kinetics was analyzed using Biacore T100 (GEHealthcare). The SR344-antibody interaction was measured by immobilizingappropriate amounts of anti-IgG (γ-chain specific) F(ab')₂ onto a sensorchip by amine coupling method, binding antibodies of interest onto thechip at pH7.4, and then running IL-6 receptor SR344 adjusted to bevarious concentrations at pH7.4 over the chip as an analyte. Allmeasurements were carried out at 37° C. The kinetic parameters,association rate constant k_(a) (1/Ms) and dissociation rate constantk_(d) (1/s) were calculated from the sensorgrams obtained bymeasurement. Then, K_(D) (M) was determined based on the rate constants.The respective parameters were determined using Biacore T100 EvaluationSoftware (GE Healthcare).

PK/PD Test to Determine the Plasma Concentrations of Antibodies, CRP,and Free Soluble IL-6 Receptor in Monkeys

The plasma concentrations in cynomolgus monkeys were determined by ELISAusing a method known to those skilled in the art.

The concentration of CRP was determined with an automated analyzer(TBA-120FR; Toshiba Medical Systems Co.) using Cias R CRP (KANTOCHEMICAL CO., INC.).

The plasma concentration of free soluble cynomolgus monkey IL-6 receptorin cynomolgus monkeys was determined by the procedure described below.All IgG antibodies (cynomolgus monkey IgG, anti-human IL-6 receptorantibody, and anti-human IL-6 receptor antibody-soluble cynomolgusmonkey IL-6 receptor complex) in the plasma were adsorbed onto Protein Aby loading 30 μl of cynomolgus monkey plasma onto an appropriate amountof rProtein A Sepharose Fast Flow resin (GE Healthcare) dried in a0.22-μm filter cup (Millipore). Then, the solution in cup was spinneddown using a high-speed centrifuge to collect the solution that passedthrough. The solution that passed through does not contain ProteinA-bound anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6receptor complex. Therefore, the concentration of free soluble IL-6receptor can be determined by measuring the concentration of solublecynomolgus monkey IL-6 receptor in the solution that passed throughProtein A. The concentration of soluble cynomolgus monkey IL-6 receptorwas determined using a method known to those skilled in the art formeasuring the concentrations of soluble human IL-6 receptor. Solublecynomolgus monkey IL-6 receptor (cIL-6R) prepared as described above wasused as a standard.

Then the percentage of soluble IL-6 receptor neutralization wascalculated by following formula.

$\frac{\begin{matrix}{{Free}\mspace{14mu}{soluble}\mspace{14mu}{IL}\text{-}6\mspace{14mu}{receptor}} \\{{concentration}\mspace{14mu}{after}\mspace{14mu}{antibody}\mspace{14mu}{administration}}\end{matrix}}{\begin{matrix}{{Soluble}\mspace{14mu}{IL}\text{-}6\mspace{14mu}{receptor}} \\{{concentration}\mspace{14mu}{before}\mspace{14mu}{antibody}\mspace{14mu}{administration}}\end{matrix}} \times 100$

INDUSTRIAL APPLICABILITY

The present invention provides antibody constant regions suitable forpharmaceuticals, whose physicochemical properties (stability andhomogeneity), immunogenicity, safety, and pharmacokinetics have beenimproved by amino acid alteration.

The invention claimed is:
 1. An IgG4 antibody comprising twopolypeptides that are antibody heavy chains, each of which comprises anidentical heavy chain constant region comprising the amino acid sequenceof SEQ ID NO: 3 with only the following amino acid changes: (i)deletions of the amino acids at EU numbering positions 446 and 447; (ii)optionally an amino acid substitution, relative to SEQ ID NO: 3, at oneor more of the following EU numbering positions: 131, 133, 137, 138,214, 217, 219, 220, 221, 222, 233, 234, 235 and 409; and (iii)optionally a deletion of the amino acid at EU numbering position 236,wherein the amino acid at EU numbering position 445 is not amidated andis at the C-terminus of each of the two polypeptides.
 2. The IgG4antibody of claim 1, wherein the amino acids at EU numbering positions233, 234, and 235 are substituted relative to the sequence of SEQ ID NO:3, and the amino acid at EU numbering position 236 is deleted.
 3. TheIgG4 antibody of claim 1, wherein the amino acids at EU numberingpositions 131, 133, 137, 138, 214, 217, 219, 220, 221, and 222 aresubstituted relative to the sequence of SEQ ID NO:
 3. 4. The IgG4antibody of claim 1, wherein the amino acid at EU numbering position 409is substituted relative to the sequence of SEQ ID NO:
 3. 5. The IgG4antibody of claim 1, wherein the amino acids at EU numbering positions131, 133, 137, 138, 214, 217, 219, 220, 221, 222, 233, 234, 235, and 409are substituted relative to the sequence of SEQ ID NO: 3, and the aminoacid at EU numbering position 236 is deleted.
 6. A pharmaceuticalcomposition comprising the antibody of claim
 1. 7. An IgG4 antibodycomprising two polypeptides that are antibody heavy chains, each ofwhich comprises an identical heavy chain constant region comprising theamino acid sequence of SEQ ID NO: 3 with only the following amino acidchanges: (i) deletions of the amino acids at EU numbering positions 446and 447; (ii) one or more substitutions relative to the sequence of SEQID NO: 3, including a substitution at one or more of the following EUnumbering positions: 131, 133, 137, 138, 214, 217, 219, 220, 221, 222,233, 234, 235 and 409; and (iii) optionally a deletion of the amino acidat EU numbering position 236, wherein the amino acid at EU numberingposition 445 is the C-terminal amino acid of each of the twopolypeptides and is not amidated.
 8. The IgG4 antibody of claim 7,wherein the amino acids at EU numbering positions 233, 234, and 235 aresubstituted relative to the sequence of SEQ ID NO: 3, and the amino acidat EU numbering position 236 is deleted.
 9. The IgG4 antibody of claim7, wherein the amino acids at EU numbering positions 131, 133, 137, 138,214, 217, 219, 220, 221, and 222 are substituted relative to thesequence of SEQ ID NO:
 3. 10. The IgG4 antibody of claim 7, wherein theamino acid at EU numbering position 409 is substituted relative to thesequence of SEQ ID NO:
 3. 11. The IgG4 antibody of claim 7, wherein theamino acids at EU numbering positions 131, 133, 137, 138, 214, 217, 219,220, 221, 222, 233, 234, 235, and 409 are substituted relative to thesequence of SEQ ID NO: 3, and the amino acid at EU numbering position236 is deleted.
 12. A pharmaceutical composition comprising the antibodyof claim
 7. 13. An IgG4 antibody comprising two polypeptides that areantibody heavy chains, each of which comprises an identical heavy chainconstant region comprising the amino acid sequence of SEQ ID NO: 3 withchanges including: deletions of the amino acids at EU numberingpositions 446 and 447; and (ii) at least one additional change at any ofEU numbering positions 118-445 in the amino acid sequence of SEQ ID NO:3, each additional change being independently selected from the groupconsisting of substitution, deletion, and insertion, wherein the atleast one additional change includes a change selected from the groupconsisting of: an amino acid substitution at EU numbering position 131,an amino acid substitution at EU numbering position 133, an amino acidsubstitution at EU numbering position 137, an amino acid substitution atEU numbering position 138, an amino acid substitution at EU numberingposition 214, an amino acid substitution at EU numbering position 217,an amino acid substitution at EU numbering position 219, an amino acidsubstitution at EU numbering position 220, an amino acid substitution atEU numbering position 221, an amino acid substitution at EU numberingposition 222, an amino acid substitution at EU numbering position 233,an amino acid substitution at EU numbering position 234, an amino acidsubstitution at EU numbering position 235, an amino acid substitution atEU numbering position 409, and a deletion of the amino acid at EUnumbering position 236, wherein the amino acid at EU numbering position445 may be substituted but is not deleted, is the C-terminal amino acidof each of the two polypeptides, and is not amidated.
 14. The IgG4antibody of claim 13, wherein the amino acids at EU numbering positions233, 234, and 235 are substituted relative to the sequence of SEQ ID NO:3, and the amino acid at EU numbering position 236 is deleted.
 15. TheIgG4 antibody of claim 13, wherein the amino acids at EU numberingpositions 131, 133, 137, 138, 214, 217, 219, 220, 221, and 222 aresubstituted relative to the sequence of SEQ ID NO:
 3. 16. The IgG4antibody of claim 13, wherein the amino acid at EU numbering position409 is substituted relative to the sequence of SEQ ID NO:
 3. 17. TheIgG4 antibody of claim 13, wherein the amino acid at EU numberingposition 236 is deleted.
 18. A pharmaceutical composition comprising theantibody of claim
 13. 19. A method of producing an IgG4 antibodycomprising two identical modified human IgG4 heavy chain constantregions with reduced C-terminal heterogeneity, the method comprising:(a) preparing nucleic acid encoding the IgG4 antibody of claim 1; (b)introducing the nucleic acid in a host cell; (c) culturing the host cellto express the nucleic acid; and (d) collecting the IgG4 antibody fromthe host cell, wherein C-terminal heterogeneity of the IgG4 antibody ina composition comprising multiple copies of the IgG4 antibody is reducedcompared to C-terminal heterogeneity in a composition comprisingmultiple copies of a naturally occurring human IgG4.
 20. A method ofproducing an IgG4 antibody comprising two identical modified human IgG4heavy chain constant regions with reduced C-terminal heterogeneity, themethod comprising: (a) preparing nucleic acid encoding the IgG4 antibodyof claim 7; (b) introducing the nucleic acid in a host cell; (c)culturing the host cell to express the nucleic acid; and (d) collectingthe IgG4 antibody from the host cell, wherein C-terminal heterogeneityof the IgG4 antibody in a composition comprising multiple copies of theIgG4 antibody is reduced compared to C-terminal heterogeneity in acomposition comprising multiple copies of a naturally occurring humanIgG4.
 21. A method of producing an IgG4 antibody comprising twoidentical modified human IgG4 heavy chain constant regions with reducedC-terminal heterogeneity, the method comprising: (a) preparing nucleicacid encoding the IgG4 antibody of claim 13; (b) introducing the nucleicacid in a host cell; (c) culturing the host cell to express the nucleicacid; and (d) collecting the IgG4 antibody from the host cell, whereinC-terminal heterogeneity of the IgG4 antibody in a compositioncomprising multiple copies of the IgG4 antibody is reduced compared toC-terminal heterogeneity in a composition comprising multiple copies ofa naturally occurring human IgG4.